Chromomycin A3 suppresses cholangiocarcinoma growth by induction of S phase cell cycle arrest and suppression of Sp1­related anti­apoptotic proteins.

Cholangiocarcinoma (CCA) is a cancer of biliary epithelium. Late diagnosis and resistance to conventional chemotherapy are the major obstacles in CCA treatment. Increased expression of anti­apoptotic proteins are observed in CCA, which might confer chemoresistance. Thus, modulations of anti­apoptotic proteins leading to apoptotic induction is the focus of this study. Chromomycin A3 (CMA3), an anthraquinone glycoside­mithramycin A analog, was selected. CMA3 strongly binds to GC­rich regions in DNA, where specificity protein 1 (Sp1), a common transcription factor of apoptosis­related proteins, is preferentially bounded. The effects of CMA3 on anti­proliferation, cell cycle arrest and apoptosis induction in CCA cells were demonstrated by MTT assay, flow cytometry and western blot analysis. The results showed CMA3 suppressed cell proliferation in vitro in the nM range. At low doses, CMA3 inhibited cell cycle progression at S phase, while it promoted caspase­dependent apoptosis at higher doses. CMA3 induced effects of apoptosis were through the suppression of Sp1­related anti­apoptotic proteins, FADD­like IL­1ß­converting enzyme­inhibitory protein, myeloid cell leukemia­1, X­linked inhibitor of apoptosis protein, cellular inhibitor of apoptosis and survivin. The anti­CCA effects of CMA3 were confirmed in the xenograft mouse model. CMA3 retarded xenograft tumor growth. Taken together, CMA3 induced apoptosis in CCA cells by diminishing the Sp1­related anti­apoptotic proteins is demonstrated. CMA3 might be useful as a chemosensitizing agent.

CoII(Chromomycin)2 Complex Induces a Conformational Change of CCG Repeats from i-Motif to Base-Extruded DNA Duplex.

We have reported the propensity of a DNA sequence containing CCG repeats to form a stable i-motif tetraplex structure in the absence of ligands. Here we show that an i-motif DNA sequence may transition to a base-extruded duplex structure with a GGCC tetranucleotide tract when bound to the (CoII)-mediated dimer of chromomycin A3, CoII(Chro)2. Biophysical experiments reveal that CCG trinucleotide repeats provide favorable binding sites for CoII(Chro)2. In addition, water hydration and divalent metal ion (CoII) interactions also play a crucial role in the stabilization of CCG trinucleotide repeats (TNRs). Our data furnish useful structural information for the design of novel therapeutic strategies to treat neurological diseases caused by repeat expansions.

Absence of Peroxiredoxin 6 Amplifies the Effect of Oxidant Stress on Mobility and SCSA/CMA3 Defined Chromatin Quality and Impairs Fertilizing Ability of Mouse Spermatozoa.

Oxidative stress, the imbalance between reactive oxygen species production and antioxidant defenses, is associated with male infertility. Peroxiredoxins (PRDXs) are antioxidant enzymes with a wide distribution in spermatozoa. PRDX6 is highly abundant and located in all subcellular compartments of the spermatozoon. Infertile men have lower levels of sperm PRDX6 associated with low sperm motility and high DNA damage. In order to better understand the role of PRDX6 in male reproduction, the aim of this study was to elucidate the impact of the lack of PRDX6 on male mouse fertility. Spermatozoa lacking PRDX6 showed significantly increased levels of cellular oxidative damage evidenced by high levels of lipid peroxidation, 8-hydroxy-deoxyguanosine (DNA oxidation), and protein oxidation (S-glutathionylation and carbonylation), lower sperm chromatin quality (high DNA fragmentation and low DNA compaction, due to low levels of protamination and a high percentage of free thiols), along with decreased sperm motility and impairment of capacitation as compared with wild-type (WT) spermatozoa. These manifestations of damage are exacerbated by tert-butyl hydroperoxide treatment in vivo. While WT males partially recovered the quality of their spermatozoa (in terms of motility and sperm DNA integrity), Prdx6(-/-) males showed higher levels of sperm damage (lower motility and chromatin integrity) 6 mo after the end of treatment. In conclusion, Prdx6(-/-) males are more vulnerable to oxidative stress than WT males, resulting in impairment of sperm quality and ability to fertilize the oocyte, compatible with the subfertility phenotype observed in these knockout mice.

Chromomycins A2 and A3 from marine actinomycetes with TRAIL resistance-overcoming and Wnt signal inhibitory activities.

A biological screening study of an actinomycetes strain assembly was conducted using a cell-based cytotoxicity assay. The CKK1019 strain was isolated from a sea sand sample. Cytotoxicity-guided fractionation of the CKK1019 strain culture broth, which exhibited cytotoxicity, led to the isolation of chromomycins A2 (1) and A3 (2). 1 and 2 showed potent cytotoxicity against the human gastric adenocarcinoma (AGS) cell line (IC50 1; 1.7 and 2; 22.1 nM), as well as strong inhibitory effects against TCF/ß-catenin transcription (IC50 1; 1.8 and 2; 15.9 nM). 2 showed the ability to overcome tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance. To the best of our knowledge, the effects of chromomycins A2 (1) and A3 (2) on TRAIL resistance-overcoming activity, and on the Wnt signaling pathway, have not been reported previously. Thus, 1 and 2 warrant potential drug lead studies in relation to TRAIL-resistant and Wnt signal-related diseases and offer potentially useful chemical probes for investigating TRAIL resistance and the Wnt signaling pathway.

High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

The crucial role of divalent metal ions in the DNA-acting efficacy and inhibition of the transcription of dimeric chromomycin A3.

Chromomycin A3 (Chro) is capable of forming a stable dimeric complex via chelation with Ni(II), Fe(II) and Co(II). According to the circular dichroism study, the dimer conformations are significantly different among the Fe(II)-, Co(II)-, and Ni(II)-containing dimeric Chro complexes; however, the dimer conformations were preserved at high temperatures. Furthermore, we conducted a systematic study to determine the effects of these divalent metal ions on the DNA-acting efficacy of dimeric Chro, including its DNA-binding affinity, DNA stabilization capacity, DNA cleavage activity, and the inhibition of transcription both in vitro and within cells. Kinetic analyses using surface plasmon resonance (SPR) showed that Ni(II)(Chro)(2) exhibited the highest K(a) with a value of 1.26 × 10(7) M(-1), which is approximately 1.6- and 3.7-fold higher than the K(a) values obtained for Co(II)(Chro)(2) and Fe(II)(Chro)(2), respectively. The T(m) and ΔG values for the DNA duplex increased after the addition of drug complexes in the following order: Ni(II)(Chro)(2)>Co(II)(Chro)(2)>Fe(II)(Chro)(2). In the DNA integrity assays, the DNA cleavage rate of Co(II)(Chro)(2) (1.2 × 10(-3) s(-1)) is higher than those of Fe(II)(Chro)(2) and Ni(II)(Chro)(2), which were calculated to be 1 × 10(-4) and 3.1 × 10(-4) s(-1), respectively. Consistent with the SPR and UV melting results, Ni(II)(Chro)(2) possesses the highest inhibitory effect on in vitro transcription and c-myc transcription within cells compared to Co(II)(Chro)(2) and Fe(II)(Chro)(2). By comparing the cytotoxicity among Co(II)(Chro)(2), Fe(II)(Chro)(2), and Ni(II)(Chro)(2) to several cancer cell lines, our studies concluded that Ni(II)(Chro)(2) displayed more potential antitumor activities than Co(II)(Chro)(2) and Fe(II)(Chro)(2) did due to its higher DNA-acting efficacy. Changes to the divalent metal ions in the dimeric Chro complexes have been correlated with improved anticancer profiles. The availability of new metal derivatives of Chro may introduce new possibilities for exploiting the unique properties of this class of compounds for therapeutic applications.

Effects of DNA minor groove binding agents on global gene expression.

The capacity of two minor groove binding agents that differ in their DNA sequence selectivity to modulate gene expression in human leukaemia cells was investigated. The chosen compounds were the chromomycin A3, a GC selective minor groove binder, and alkamin, an AT selective minor groove binder. As revealed by DNA microarray analysis of 6000 genes, at equitoxic doses, 5×IC(50) values for growth inhibition, the two drugs disturbed transcription, resulting in both up- and down-regulation of many hundreds of genes, 24 h after drug exposure. Direct comparisons between the most affected genes and also the cluster analysis indicated a relatively low degree of similarity between the tow expression profiles. Moreover, the ontological and the pathway responses also indicated a distinguished biological responses. Chromomycin treatment was characterized by many negative impacts on the important cellular functions and by the activation for those functions that usually take the cells towards apoptosis. In the second biological profile, the domination of many positive functions might indicate that the cells were attempting to overcome and repair the alkamin assault. Examples of these functions are positive regulation of gene expression, positive regulation of macromolecule biosynthetic processes, the cell cycle pathway and DNA repair.

Differential inhibition of restriction enzyme cleavage by chromophore-modified analogues of the antitumour antibiotics mithramycin and chromomycin reveals structure-activity relationships.

Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A(3) (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles.

Inhibition of a Zn(II)-containing enzyme, alcohol dehydrogenase, by anticancer antibiotics, mithramycin and chromomycin A3.

One of the major attributes for the biological action of the aureolic acid anticancer antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) is their ability to bind bivalent cations such as Mg(II) and Zn(II) ions and form high affinity 2:1 complexes in terms of the antibiotic and the metal ion, respectively. As most of the cellular Zn(II) ion is found to be associated with proteins, we have examined the effect of MTR/CHR on the structure and function of a representative structurally well characterized Zn(II) metalloenzyme, alcohol dehydrogenase (ADH) from yeast. MTR and CHR inhibit enzyme activity of ADH with inhibitory constants of micromolar order. Results from size-exclusion column chromatography, dynamic light scattering, and isothermal titration calorimetry have suggested that the mechanism of inhibition of the metalloenzyme by the antibiotics is due to the antibiotic-induced disruption of the enzyme quaternary structure. The nature of the enzyme inhibition, the binding stoichiometry of two antibiotics per monomer, and comparable dissociation constants for the antibiotic and free (or substrate-bound) ADH imply that the association occurs as a consequence of the binding of the antibiotics to Zn(II) ion present at the structural center. Confocal microscopy shows the colocalization of the antibiotic and the metalloenzyme in HepG2 cells, thereby supporting the proposition of physical association between the antibiotic(s) and the enzyme inside the cell.

Studies of sequence-specific DNA binding, DNA cleavage, and topoisomerase I inhibition by the dimeric chromomycin A3 complexed with Fe(II).

Chromomycin A3 (Chro) has been evidenced to exhibit much higher binding affinity toward Fe(II) by forming a highly stable 2:1 drug/metal complex, compared to its structural analogue, mithramycin (Mith). Different properties of the [(Chro)2-Fe(II)] complex acting on DNA, such as sequence specificity, DNA cleavage, and topoisomerase I (TopI) inhibition were studied. Kinetic analyses of surface plasmon resonance showed that the affinity of the [(Chro)2-Fe(II)] complex upon binding to hairpin DNA duplexes containing various tetranucleotide sequences follows the order: GGCC > CGCG > CCGG approximately GCGC > AGCT > ACGT > TGCA > TCGA. According to circular dichroism (CD) studies, most hairpin DNA duplexes appeared to retain their B-type conformations in the presence of the [(Chro)2-Fe(II)] complex, except the duplex containing the GGCC sequence, which exhibited the features of both A- and B-type DNA. In DNA-cleavage assays, the [(Chro) 2-Fe(II)] complex was shown to cause single-stranded cleavage of plasmid DNA because of a Fenton-type reaction. DNA cleavage activity of the [(Chro) 2-Fe(II)] complex was increased at low pH. Moreover, the complex was capable of inhibiting TopI activity. The [(Chro)2-Fe(II)] complex exhibited higher cytotoxicity than the [(Mith) 2-Fe(II)] complex in several cancer cell lines, most likely owing to its more stable dimeric structure and higher DNA-binding affinity. Our results provide significant evidence that the [(Chro)2-Fe(II)] complex could be promising in terms of its biological applications in the future.

Involvement of a chromomycin ABC transporter system in secretion of a deacetylated precursor during chromomycin biosynthesis.

Chromomycin A(3) is an antitumour antibiotic that acts by inhibiting transcription and replication of DNA. The producer micro-organism Streptomyces griseus subsp. griseus is highly resistant to chromomycin A(3) and to the structurally related compound mithramycin upon induction with chromomycin A(3). The biosynthetic gene cluster of chromomycin contains three genes involved in self-resistance to chromomycin in S. griseus: cmrA and cmrB encode a type I ATP-binding cassette (ABC) transporter, and cmrX encodes a UvrA-like protein of ABC excision nuclease systems. These genes are linked in the chromosome, together with a gene encoding a transcriptional repressor (cmmRII). Involvement of these genes in chromomycin resistance was determined through gene inactivation, and heterologous expression in Streptomyces albus. Inactivation of cmrX produced a chromomycin-sensitive low-producer strain, while inactivation of cmmRII generated a high-chromomycin-producer strain, which was resistant to chromomycin, and also to mithramycin. Expression of either cmrA and cmrB, or cmrX, in S. albus generated strains with low chromomycin resistance; it was therefore necessary to co-express the three genes to achieve high levels of resistance. However, the CmrAB ABC transporter conferred a high level of resistance to the biosynthesis intermediate 4A,4E-O-dideacetyl-chromomycin A(3). A model is proposed for the biosynthesis of, and self-resistance to, chromomycin A(3) in S. griseus subsp. griseus.

GC-rich heterochromatin in silver stained nucleolar organizer regions (NORs) fluoresces with Chromomycin A3 (CMA3) staining in three species of teleostean fishes (Pisces).

In a bid to ascertain the molecular architecture of the silver positive regions (NORs) in chromosomes of three species of fish, namely, Hemibagrus menoda (Hamilton), Sperata seenghala (Sykes) (Fam: Bagridae) and Mastacembelus armatus (Lacep6de) (Fam: Mastacembelidae), an additional staining methodology using a fluorochrome dye (Chromomycin A3) was deployed along with the AgNO3 technique. The nucleolar organizing regions (NORs) were located terminally at the shorter arms (Tp) of one pair of submetacentric chromosomes (No.3) in H. menoda (2n=58), at the longer arms (Tq) of one pair of submetacentric chromosomes (No.5) in S. seenghala (2n=50) and at the shorter arm (Tp) of one pair of homologous submetacentric chromosomes (No.6) in M. armatus (2n=48). Staining with Chromomycin A3 produced bright fluorescing zones in GC-rich heterochromatin of Ag-positive NORs. The results indicate a more general trend of existence of an overlapping region between NOR and GC-rich fluorescing zones, the active sites of rRNA genes (rDNA) in this primitive group of vertebrates although exceptions to this situation has been reported in a couple of extant fish species earlier. More data utilizing such combined methodologies are warranted to understand the structural organization of fish chromosomes more precisely.

Deoxysugar transfer during chromomycin A3 biosynthesis in Streptomyces griseus subsp. griseus: new derivatives with antitumor activity.

Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-D-oliose (sugar A) and 4-O-methyl-D-oliose (sugar B), and a trisaccharide of D-olivose (sugar C), D-olivose (sugar D), and 4-O-acetyl-L-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two D-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.

DNA structural similarity in the 2:1 complexes of the antitumor drugs trabectedin (Yondelis) and chromomycin A3 with an oligonucleotide sequence containing two adjacent TGG binding sites on opposing strands.

Yondelis (trabectedin) is an antitumor ecteinascidin that binds covalently to the 2-amino group of the central guanine in the minor groove of selected DNA pyrimidine-G-G and purine-G-C triplets. Chromomycin A3 is an aureolic acid derivative that binds noncovalently to the DNA minor groove in G/C-rich triplet sites as a metal-chelated dimer. Despite their different binding modes, the cytotoxicity profiles of these two drugs, as assessed in the COMPARE analysis carried out by the National Cancer Institute on data from 60 human tumor cell lines, are highly correlated (Pearson's correlation coefficient of 0.96). We now report that in an oligonucleotide containing the "natural bending element" TGGCCA, the structural distortions inflicted by the tail-to-tail bonding of two trabectedin molecules to adjacent target sites on opposing strands are strikingly similar to those observed in a crystal containing d(TTGGCCAA)2 and two bound chromomycin A3 molecules arranged in a head-to-tail orientation in the minor groove. In both complexes, the double helix is characterized by being considerably unwound and possessing a notably widened minor groove. Binding of the drugs to this sequence could be favored by the distinct bends at each of the TpG steps that are already present in the free oligonucleotide. Simultaneous drug binding to the two strands in the manner described here is proposed to stabilize the helical structure of duplex DNA to prevent or hamper strand separation and stall replication and transcription forks.

Human cervical mucus can act in vitro as a selective barrier against spermatozoa carrying fragmented DNA and chromatin structural abnormalities.

PURPOSE: We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. METHODS: Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and "endogenous" nick translation. RESULTS: The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. CONCLUSIONS: We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.

Chromomycin A3 inhibits influenza a virus multiplication in chick embryo fibroblast cells.

The multiplication of Ulster 73 virus, an avian strain of type A influenza virus, was blocked in chick embryo fibroblast cells, CEF, by treatment with 0.5 microg/ml of chromomycin A3 whereas in LLC-MK2 cells no inhibition of replication was observed. Virus-induced polypeptide synthesis in chick embryo fibroblast cells was confined to the synthesis of PB2, PB1 and PA subunits of the RNA dependent-RNA polymerase, the nucleoprotein NP, the non-structural protein NS1, the haemagglutinin HA, the non-structural protein NS2; only the membrane M1 polypeptide synthesis was greatly inhibited. Viral unpolyadenylated cRNAs synthesis was studied at a late time of the infection, 8 hours p.i.: chromomycin A3 was able to inhibit the "novo" synthesis of complementary RNA poly(A)- and segment 7 of virion RNA. The mode of action of the drug in chick embryo fibroblast cells is discussed.

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+).

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.

Role of Mg2+ in the interaction of anticancer antibiotic, chromomycin A3 with DNA: does neutral antibiotic bind DNA in absence of the metal ion?

Antitumor antibiotic, Chromomycin A3 (CHR), inhibits DNA replication and transcription via reversible interaction with double stranded DNA with GC-base specificity. The interaction, at and above physiological pH, requires the presence of bivalent metal ions, such as Mg2+. Anionic antibiotic does not bind DNA in the absence of Mg2+. In this paper we have examined the structural potential of neutral CHR at pH 5.2 to bind DNA in the absence of Mg2+. We have demonstrated the ability of the neutral antibiotic to bind DNA by means of different spectroscopic techniques and evaluated the necessary thermodynamic parameters for elucidation of the molecular basis of recognition. The results are compared with the scenario when Mg2+ is present in the system, because the ultimate aim of these studies is to elucidate the role of Mg2+ in CHR-DNA recognition. Neutral CHR binds to Mg2+ with lesser affinity than its anionic form. Spectroscopic features of the drug and its Mg2+ complex indicate self association of the antibiotic in the absence and presence of Mg2+. GC-base specificity of the drug and its Mg2+ complex are retained at pH 5.2, though the modes of recognition of DNA by the two ligands are different. Minor groove width of DNA plays a role in the accommodation of the ligand(s) during the GC base specific recognition while positive charge of Mg2+ in CHR:Mg2+ complex further facilitates the association. Relatively lower affinity of the neutral drug and its Mg2+ complex for DNA can be ascribed to the self association of these ligands in the absence of DNA.

Evaluation of the effectiveness of DNA-binding drugs to inhibit transcription using the c-fos serum response element as a target.

Previous work has demonstrated that sequence-selective DNA-binding drugs can inhibit transcription factors from binding to their target sites on gene promoters. In this study, the potency and effectiveness of DNA-binding drugs to inhibit transcription were assessed using the c-fos promoter's serum response element (SRE) as a target. The drugs chosen for analysis included the minor groove binding agents chromomycin A(3) and Hoechst 33342, which bind to G/C-rich and A/T-rich regions, respectively, and the intercalating agent nogalamycin, which binds G/C-rich sequences in the major groove. The transcription factors targeted, Elk-1 and serum response factor (SRF), form a ternary complex (TC) on the SRE that is necessary and sufficient for induction of c-fos by serum. The drugs' abilities to prevent TC formation on the SRE in vitro were nogalamycin > Hoechst 33342 > chromomycin. Their potencies in inhibiting cell-free transcription and endogenous c-fos expression in NIH3T3 cells, however, were chromomycin > nogalamycin > Hoechst 33342. The latter order of potency was also obtained for the drugs' cytotoxicity and inhibition of general transcription as measured by [(3)H]uridine incorporation. These systematic analyses provide insight into how drug and transcription factor binding characteristics are related to drugs' effectiveness in inhibiting gene expression.

Production and antiproliferative activity of liposomes containing the antitumour drug chromomycin A3.

In the present paper the production and characterization of liposomes are described as a specialized drug delivery system for chromomycin. Liposomes were prepared by the reverse phase evaporation technique followed by extrusion through polycarbonate filters; afterwards the vesicles were characterized in terms of dimensions, morphology and encapsulation efficacy. The aim of this work was to produce a drug delivery system able to reduce the toxicity problems related to the administration of this drug. The analysis of the in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that ionic and neutral liposomes containing chromomycin are 1.5 and 7-fold more effective respectively as compared to the free drug. Based on these results and taking into account the increased solubility of the drug in this system, liposomes could represent a promising drug delivery system for use in the experimental therapy using chromomycin.