Inhibitory effects of anthracyclines on partially purified 5'-3' DNA helicase of Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites. METHODS: In this study, a P. falciparum 5' to 3' DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind 32P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated. RESULTS: The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3' or 5' end and a linear substrate with 3' end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC50 value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC50 = 0.1 ± 0.002 µM). CONCLUSION: Partial purification and characterization of 5'-3' DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3' to 5' DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.

Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells.

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48 h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214-0.242 µM) than in MDA MB 231 (IC50: 0.346-0.37 µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68-41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

In silico identification and in vitro validation of nogalamycin N-oxide (NSC116555) as a potent anticancer compound against non-small-cell lung cancer cells.

The epidermal growth factor receptor (EGFR) was found to be overexpressed in several cancers, especially in lung cancers. Finding new effective drug against EGFR is the key to cancer treatment. In this study, the GOLD docking algorithm was used to virtually screen for novel human EGFR inhibitors from the NCI database. Thirty-four hit compounds were tested for EGFR-tyrosine kinase (TK) inhibition. Two potent compounds, 1-amino-4-(4-[4-amino-2-sulfophenyl]anilino)-9,10-dioxoanthracene-2-sulfonic acid (NSC125910), and nogalamycin N-oxide (NSC116555) were identified with IC50 values against EGFR-TK comparable to gefitinib; 16.14 and 37.71 nM, respectively. However, only NSC116555 demonstrated cytotoxic effects against non-small-cell lung cancer, A549, shown in the cell cytotoxicity assay with an IC50 of 0.19 + 0.01 µM, which was more potent than gefitinib. Furthermore, NSC116555 showed cytotoxicity against A549 via apoptosis in a dose-dependent manner.

ATPase activity of Plasmodium falciparum MLH is inhibited by DNA-interacting ligands and dsRNAs of MLH along with UvrD curtail malaria parasite growth.

Malaria caused by Plasmodium falciparum is the major disease burden all over the world. Recently, the situation has deteriorated because the malarial parasites are becoming progressively more resistant to numerous commonly used antimalarial drugs. Thus, there is a critical requirement to find other means to restrict and eliminate malaria. The mismatch repair (MMR) machinery of parasite is quite unique in several ways, and it can be exploited for finding new drug targets. MutL homolog (MLH) is one of the major components of MMR machinery, and along with UvrD, it helps in unwinding the DNA. We have screened several DNA-interacting ligands for their effect on intrinsic ATPase activity of PfMLH protein. This screening suggested that several ligands such as daunorubicin, etoposide, ethidium bromide, netropsin, and nogalamycin are inhibitors of the ATPase activity of PfMLH, and their apparent IC50 values range from 2.1 to 9.35 µM. In the presence of nogalamycin and netropsin, the effect was significant because in their presence, the V max value dropped from 1.024 µM of hydrolyzed ATP/min to 0.596 and 0.643 µM of hydrolyzed ATP/min, respectively. The effect of double-stranded RNAs of PfMLH and PfUvrD on growth of P. falciparum 3D7 strain was studied. The parasite growth was significantly inhibited suggesting that these components belonging to MMR pathway are crucial for the survival of the parasite.

Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth.

BACKGROUND: Human malaria parasite infection and its control is a global challenge which is responsible for ~0.65 million deaths every year globally. The emergence of drug resistant malaria parasite is another challenge to fight with malaria. Enormous efforts are being made to identify suitable drug targets in order to develop newer classes of drug. Helicases play crucial roles in DNA metabolism and have been proposed as therapeutic targets for cancer therapy as well as viral and parasitic infections. Genome wide analysis revealed that Plasmodium falciparum possesses UvrD helicase, which is absent in the human host. RESULTS: Recently the biochemical characterization of P. falciparum UvrD helicase revealed that N-terminal UvrD (PfUDN) hydrolyses ATP, translocates in 3' to 5' direction and interacts with MLH to modulate each other's activity. In this follow up study, further characterization of P. falciparum UvrD helicase is presented. Here, we screened the effect of various DNA interacting compounds on the ATPase and helicase activity of PfUDN. This study resulted into the identification of daunorubicin (daunomycin), netropsin, nogalamycin, and ethidium bromide as the potential inhibitor molecules for the biochemical activities of PfUDN with IC50 values ranging from ~3.0 to ~5.0 µM. Interestingly etoposide did not inhibit the ATPase activity but considerable inhibition of unwinding activity was observed at 20 µM. Further study for analyzing the importance of PfUvrD enzyme in parasite growth revealed that PfUvrD is crucial/important for its growth ex-vivo. CONCLUSIONS: As PfUvrD is absent in human hence on the basis of this study we propose PfUvrD as suitable drug target to control malaria. Some of the PfUvrD inhibitors identified in the present study can be utilized to further design novel and specific inhibitor molecules.

Personalized chemotherapy profiling using cancer cell lines from selectable mice.

PURPOSE: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. EXPERIMENTAL DESIGN: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. RESULTS: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)-approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. CONCLUSION: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.

Identification of late-stage glycosylation steps in the biosynthetic pathway of the anthracycline nogalamycin.

Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C-C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C-C bond intact (nogalamycin R) or by 2-deoxyfucose without the C-C bond (nogalamycin F). In addition, a monoglycosylated intermediate-3',4'-demethoxynogalose-1-hydroxynogalamycinone-was isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the cluster-snogZ-appears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II.

Structural effects of nogalamycin, an antibiotic antitumour agent, on DNA.

The structural changes of DNA, induced by the antitumour antibiotic nogalamycin, have been studied by atomic force microscopy (AFM). The transformation in the tertiary structure of 4361bp long plasmid pBR322 DNA, after incubation with nogalamycin at 37 degrees C, has been monitored at the single molecule level. The AFM topographs of free DNA and the DNA-nogalamycin complex, incubated for 6, 12, 24, 36 and 48h, reveal a gradual change from the circular supercoiled form having strand crossovers to the more compact plectonemic superhelix. With increasing incubation time, the extent of plectonemic coiling increases, indicating increasing level of drug binding via intercalative mode. Supportive evidences are obtained from the CD and UV-vis spectroscopic studies. To our knowledge, this is the first report on an AFM imaging study of the effects of nogalamycin, an anthracyclin intercalator, on DNA.

Complexation of anthracycline drugs with DNA in the presence of caffeine.

The competitive binding of anthracycline antitumour drugs, [daunomycin (DAU), doxorubicin (DOX) or nogalamycin (NOG)], with caffeine (CAF) to a model DNA oligomer has been investigated by 500 MHz 1H NMR spectroscopy under physiological solution conditions. The method depends on the stepwise analysis of one-component (self-association), two-component (hetero-association and DNA complexation) and three-component interactions, in order to de-convolute the overall binding of the anthracycline antibiotic and CAF to DNA into two competing processes, viz. hetero-association of the antibiotic-CAF ('interceptor' action of CAF) and CAF-DNA complexation ('protector' action of CAF). It is found that the complexation of DAU with DNA in the presence of CAF is mainly affected by the CAF-DNA complexation, whereas the binding of either DOX or NOG to DNA is affected approximately equally by both the CAF-DNA complexation and CAF-antibiotic hetero-association. Quantitative evaluation of the three-component mixture of drug-CAF-DNA has enabled the proportion of the antibiotic displaced from DNA on addition of CAF to be calculated over a large range of CAF concentration, which may provide a quantitative basis for the change in anthracycline-related toxicity on addition of CAF.

The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5'- to 3'-direction.

Nucleolin is a major nucleolar phosphoprotein of exponentially growing eukaryotic cells. Here we report the cloning, purification, and characterization of the C-terminal glycine/arginine-rich (GAR) domain of pea nucleolin. The purified recombinant protein (17 kDa) shows ATP-/Mg(2+)-dependent DNA helicase and ssDNA-/Mg(2+)-dependent ATPase activities. The enzyme unwinds DNA in the 5'- to 3'-direction, which is the first report in plant for this directional activity. It unwinds forked/non-forked DNA with equal efficiency. The anti-nucleolin antibodies immunodepleted the activities of the enzyme. The DNA interacting ligands nogalamycin, daunorubicin, actinomycin C1, and ethidium bromide were inhibitory to DNA unwinding (with K(i) values of 0.40, 2.21, 8.0, and 9.0 microM, respectively) and ATPase (with K(i) values of 0.43, 1.65, 4.6, and 7.0 microM, respectively) activities of the enzyme. This study confirms that the unwinding and ATPase activities of pea nucleolin resided in the GAR domain. This study should make important contribution to our better understanding of DNA transaction in plants, mechanism of DNA unwinding, and the mechanism by which these ligands can disturb genome integrity.

Drug-induced stabilisation of a mismatched C-T base pair in a DNA hairpin.

We have shown that a key feature of drug binding, namely specific G-C base pair recognition at a 5'-TG step, can induce a number of novel structural features when an extrahelical base is inserted in close proximity to the drug binding site; we have clearly demonstrated the formation of a stabilised C-T mismatched base pair at a non-terminal site.

A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' direction.

A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3'-->5' direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2+-dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+) is required. The DNA intercalating agents actinomycin C1, ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of 5.6, 5.2, 4.0 and 0.71 micro Ms, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA-inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants.

Transcription factor Sp1 phosphorylation induced by shear stress inhibits membrane type 1-matrix metalloproteinase expression in endothelium.

Membrane type 1-matrix metalloproteinase (MT1-MMP) plays a key role in endothelial cell migration, matrix remodeling, and angiogenesis. Previous studies demonstrated that a mechanical force, cyclic strain, increases MT1-MMP expression by displacing Sp1 with increased Egr-1 expression and binding to the promoter site. However, the effect of shear stress (SS) on MT1-MMP expression is poorly understood. Although Egr-1 mRNA transcription and protein was induced (7.6-fold) in response to SS (n = 5, 0-8 h, p < 0.05), SS decreased MT1-MMP mRNA transcription and protein levels in a time-dependent fashion (10, 50, and 90% reduction at 1, 4, and 8 h, respectively; n = 5, p < 0.05). Egr-1 protein was increased after SS and cyclic strain, but Sp1 was serine-phosphorylated only after SS. SS increased Sp1 DNA binding (3.8-, 5.8-, and 2.4-fold increase at 1, 4, and 8 h, respectively; n = 5, p < 0.05) that was inhibitable by calf intestinal phosphatase. Thus, SS inhibits MT1-MMP expression despite Egr-1 up-regulation by inducing the serine phosphorylation of Sp1, which in turn increases its binding affinity for its site on the MT1-MMP promoter, reducing the ability of Egr-1 to displace it. These data illustrate the complex control of microvascular endothelial cell MT1-MMP expression in response to distinct environmental stimuli (cyclic strain versus shear stress), consisting of both the modulation of specific transcription factor expression (Egr-1) as well as transcription factor post-translational modification (serine phosphorylation of Sp1).

Post-treatment effects of DNA topoisomerase inhibitors on UVB- and X-ray-induced chromosomal aberration formations.

Post-treatments with nogalamycin, an inhibitor of DNA topoisomerase I, for last 3h of the culture (during the G2 phase) drastically enhanced the yield of ultraviolet light B (UVB)-induced exchange-type chromatid aberrations, while showing little effect on the formation of breakage-type aberrations in Chinese hamster V79 cells. These results are very similar to those obtained with ICRF-193, an inhibitor of topoisomerase II, with respect to the effect on UVB-induced chromatid aberrations. Thus, both types of topoisomerases may suppress the formation of exchange-type chromatid aberrations in the G2 phase which is suggested to be the principal stage of the cell cycle for chromatid aberration formation.In human lymphocytes irradiated with X-rays before phytohaemagglutinin (PHA) stimulation, post-treatments with nogalamycin through the whole cell cycle enhanced only the yield of dicentrics, while showing little effect on the yields of any other chromosome-type aberrations. Nogalamycin added 6h after PHA stimulation to irradiated cells also showed almost the same effects, whereas, addition of nogalamycin 24h after PHA stimulation showed no effect on X-ray-induced chromosome-type aberrations. These results suggest that X-ray-induced DNA damage lead to chromosome-type aberrations before the start of S phase and topoisomerase I may suppress the formation of dicentrics, exchange-type chromosome aberrations. Post-treatments with ICRF-193 showed no effect on the formation of X-ray-induced chromosome-type aberrations, suggesting nonparticipation of topoisomerase II in this process.

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents.

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

Drug recognition of a DNA single strand break: nogalamycin intercalation between coaxially stacked hairpins.

Two DNA hairpin motifs (5'-GCGAAGC-3' and 5'-ACGA AGT-3'), both stabilized by a 5'-GAA loop, have been used to design novel intramolecular double hairpin structures (5'-GCGAAGCACGAAGT-3' and 5'-ACGAAGTGCG AAGC-3') in which coaxial stacking of the two hairpin components generates a double-stranded stem region effectively with a single-strand break in the middle of the sequence at either the TG or CA step between unconnected 3' and 5' terminal bases. We have investigated by NMR the conformation and dynamics of the DNA at the strand break site. We show that mutual stacking significantly enhances the stability of each hairpin. Further, the anthracycline antibiotic nogalamycin binds cleanly to the 5'-TG (5'-CA) site formed by the mutually stacked hairpins despite the break in the sugar-phosphate backbone on one strand. The complex resembles the structure of nogalamycin-DNA complexes with the drug bound at 5'-TG sites in intact duplex sequences, with pi-stacking interactions probably the single dominant stabilizing interaction.

Sp1 phosphorylation regulates inducible expression of platelet-derived growth factor B-chain gene via atypical protein kinase C-zeta.

Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-zeta blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-zeta (on residue Thr(410)). These findings demonstrate for the first time that PKC-zeta and Sp1 phosphorylation mediate the inducible expression of this growth factor.

Human mismatch repair and G*T mismatch binding by hMutSalpha in vitro is inhibited by adriamycin, actinomycin D, and nogalamycin.

Loss of the human DNA mismatch repair pathway confers cross-resistance to structurally unrelated anticancer drugs. Examples include cisplatin, doxorubicin (adriamycin), and specific alkylating agents. We focused on defining the molecular events that link adriamycin to mismatch repair-dependent drug resistance because adriamycin, unlike drugs that covalently modify DNA, can interact reversibly with DNA. We found that adriamycin, nogalamycin, and actinomycin D comprise a class of drugs that reversibly inhibits human mismatch repair in vitro at low micromolar concentrations. The substrate DNA was not covalently modified by adriamycin treatment in a way that prevents repair, and the inhibition was independent of the number of intercalation sites separating the mismatch and the DNA nick used to direct repair, from 10 to 808 base pairs. Over the broad concentration range tested, there was no evidence for recognition of intercalated adriamycin by MutSalpha as if it were an insertion mismatch. Inhibition apparently results from the ability of the intercalated drug to prevent mismatch binding, shown using a defined mobility shift assay, which occurs at drug concentrations that inhibit repair. These data suggest that adriamycin interacts with the mismatch repair pathway through a mechanism distinct from the manner by which covalent DNA lesions are processed.

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.