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PubMed1995

Monocyte chemoattractant protein-3 is a functional ligand for CC chemokine receptors 1 and 2B.

The CC chemokine monocyte chemoattractant protein-3 (MCP-3) activates human monocytes, lymphocytes, basophils, and eosinophils. MCP-3 has been reported to induce [Ca2+]i changes in cells transfected with the monocyte-selective MCP-1 receptor 2B (CC CKR2B) and competes for 125I-MCP-1 binding on CC CKR2B, suggesting that it may mediate monocyte responses to MCP-3. However, we now show that MCP-3 is a ligand and potent agonist for the macrophage inflammatory protein-1 alpha (MIP-1 alpha)/regulated on activation, normal T expressed, and secreted protein (RANTES) receptor CC CKR1 (rank order for [Ca2+]i changes = MIP-1 alpha > MCP-3 > RANTES), which is expressed in monocytes > neutrophils > eosinophils. 125I-MCP-3 bound directly to CC CKR1 and CC CKR2B (Ki = 8 and 7 nM, respectively). Binding to CC CKR1 was competed by all CC chemokines tested except MCP-1. In contrast, binding to CC CKR2B was competed only by MCP-3 and MCP-1. Both MCP-1 and MCP-3 were equipotent agonists (EC50 = 10 nM for [Ca2+]i changes). Thus, MCP-3 is a functional ligand for both CC CKR1 and CC CKR2B, which otherwise have distinct selectivities for CC chemokines. These data suggest that monocyte responses to MCP-3 could be mediated by both CC CKR2B and CC CKR1, whereas eosinophil responses to MCP-3 could be mediated by CC CKR1.

Amino Acid Sequence↗

PubMedDate not supplied

Determination of the structural features of (+)-CC-1065 that are responsible for bending and winding of DNA.

Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity, [Hurley, L. H., Lee, C.-S., McGovern, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19 degrees, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5'-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered.

Antibiotics, Antineoplastic↗

PubMed1995

Cloning and functional expression of a human eosinophil CC chemokine receptor.

Eosinophils undergo chemotaxis, degranulate, and exhibit [C2+]i changes in response to the human CC chemokines macrophage inflammatory protein (MIP)-1 alpha, regulated on activation, normal T expressed and secreted (RANTES), and monocyte chemoattractant protein-3 (MCP-3), but the receptors involved have not been defined. We have isolated a human cDNA encoding the first eosinophil-selective chemokine receptor, designated CC chemokine receptor 3 (CC CKR3). CC CKR3 is a seven-transmembrane domain G protein-coupled receptor most closely related to the previously reported monocyte- and neutrophil-selective receptor CC CKR1 (also known as the MIP-1 alpha/RANTES receptor). When [Ca2+]i changes were monitored in stably transfected human embryonic kidney 293 cells, MIP-1 alpha and RANTES were both potent agonists for CC CKR3 and CC CKR1. However, MIP-1 beta was also an agonist for CC CKR3 but not CC CKR1; MCP-3 was an agonist for CC CKR1 but not CC CKR3. CC CKR3 may be one of the host factors responsible for selective recruitment of eosinophils to sites of inflammation.

Amino Acid Sequence↗

PubMed1995

Clara cell protein (CC-16) induces a phospholipase A2-mediated inhibition of fibroblast migration in vitro.

Clara cell protein (CC-16, also designated CC-10) is synthesized by the bronchiolar epithelium and has been suggested as an inhibitor of phospholipase A2 (PLA2) activity. Therefore, CC-16 is a candidate for controlling inflammatory events in the lung. Because CC-16 amounts and function may be altered in fibrosing lung diseases in which bronchiolar injury has been reported, it was measured in alveolar fluids and sera. Secretory PLA2 activity in alveolar fluids and the influence of CC-16 on platelet-derived growth factor-induced human fibroblast chemotaxis and cytosolic PLA2 activity were also explored. CC-16 content was decreased in alveolar fluids from idiopathic pulmonary fibrosis (IPF: 1.3 +/- 0.1 mg/L) and bleomycin lung (1.1 +/- 0.2 versus 2.1 +/- 0.2 mg/L in controls, p < 0.05), whereas there was a three- to ninefold increase in secretory PLA2 activity (p < 0.05 versus controls). CC-16 inhibited fibroblast chemotaxis in a dose-dependent manner (90% inhibition at 30 micrograms/ml CC-16). This inhibition was reversed by reducing CC-16. CC-16 was also able to lower fibroblastic cytosolic PLA2 activity by 50% in vitro. In summary, CC-16 is able to inhibit fibroblast chemotaxis in vitro by mechanisms that may be related to a blockage of cytosolic PLA2 activity. It can be postulated that CC-16 deficiency may contribute to fibroblast burden activity in fibrosing lung diseases.

Adult↗

PubMed2002

Aberrant development of Plasmodium falciparum in hemoglobin CC red cells: implications for the malaria protective effect of the homozygous state.

Although selection of hemoglobin C (HbC) by malaria has been speculated for decades, only recently have epidemiologic studies provided support for HbC protection against malaria in West Africa. A reduced risk of malaria associated with the homozygous CC state has been attributed to the inability of CC cells to support parasite multiplication in vitro. However, there have been conflicting data and conclusions regarding the ability of CC red cells to support parasite replication. Reports that parasites cannot multiply in CC cells in vitro contrast with detection of substantial parasite densities in CC patients with malaria. We have therefore investigated Plasmodium falciparum growth in CC cells in vitro. Our data show that the multiplication rate of several P falciparum lines is measurable in CC cells, but lower than that in AA (HbA-normal) cells. A high proportion of ring forms and trophozoites disintegrates within a subset of CC cells, an observation that accounts for the overall lower replication rate. In addition, knobs present on the surface of infected CC cells are fewer in number and morphologically aberrant when compared with those on AA cells. Events in malaria pathogenesis that involve remodeling of the erythrocyte surface and the display of parasite antigens may be affected by these knob abnormalities. Our data suggest that only a subset of CC cells supports normal parasite replication and that components of malaria protection associated with the CC state may affect the parasite's replication capacity and involve aberrant knob formation on CC cells.

Animals↗

PubMed1989

Interaction of CC-1065 and its analogues with mouse DNA and chromatin.

CC-1065 is a potent antitumor antibiotic which is cytotoxic to P388 and L1210 leukemia cells in vitro and in vivo. CC-1065 covalently binds to calf thymus DNA preferentially to adenine-thymine regions at N3 of adenine. Here, we compare CC-1065 interaction with P388-derived chromatin, DNA, and histones as measured by electronic absorption and circular dichroism. Two CC-1065 analogues (U-71,184 and its enantiomer, U-71,185) which show different biological activities from CC-1065 were also studied. The shape and temporal behavior of the induced circular dichroism curves generated by CC-1065 or its analogues bound to chromatin were similar to CC-1065 plus DNA. This suggested that CC-1065 and its analogues bind to the minor groove of chromatin DNA in a manner similar to calf thymus DNA. However, the binding of CC-1065 and its analogues to DNA induced a more intense circular dichroism band than binding to chromatin. The order of interaction for both chromatin and DNA was CC-1065 greater than U-71,184 greater than U-71,185. In contrast to the essentially irreversible binding to DNA after 24-h incubation, binding to chromatin was primarily a reversible interaction, the degree of reversibility being U-71,185 greater than U-71,184 = CC-1065. CC-1065 binds weakly and nonspecifically to histones.

Animals↗

PubMed1987

A circular dichroism study of the binding of CC-1065 to B and Z form poly(dl-5BrdC).poly(dl-5BrdC).

CC-1065, Benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1,6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8,8a-tetrahydro-7-methyl-4- oxocyclopropa[c]pyrrolo[3,2-e]indol-2(1H)-yl)carbonyl]benzo [1,2-b:4,3-b']dipyrrol-3(2H)-yl]carbonyl]-1,6-dihydro-4-hydroxy- 5-methoxy-, (7bR,8aS), binds to the B form of poly(dl-5BrdC).poly(dl-5BrdC) to yield a reversibly bound species whose stability with respect to an irreversibly bound species (presumably the inosine N-3 adduct) is much greater than it is for other DNA polymers. Competitive binding experiments with netropsin, show that this reversibly bound species of CC-1065 contains CC-1065 in the minor groove of the double helix. A review of the CC-1065 binding data obtained on other synthetic DNA polymers suggests that the widely different rates of species conversion shown by these polymers may result from small differences in DNA secondary structure rather than from different alkylating abilities of the adenine or inosine N-3 active site. CC-1065 converts the Z-form of poly(dl-5BrdC).poly(dl-5BrdC) in 3.5 M sodium chloride to the B form and does not bind to the Z form in this solvent system. CC-1065 bound to the B form polymer inhibits the formation of the Z form if the helix is saturated with CC-1065. Regions of the polymer without bound CC-1065 can convert to the Z form with added salt, producing a situation where the polymer contains both the B and Z conformations. In 4.0 M sodium chloride, where the Z conformation is also predominate, the addition of CC-1065 causes chiral aggregates to form, and CC-1065 binds to the aggregates. The addition of dimethylformamide in the absence of CC-1065 or a simple dilution of the 4.0 M sodium chloride polymer solution with water also causes aggregation, indicating that the Z form of this polymer in 4.0 M sodium chloride is unstable with respect to an aggregated form.

Antibiotics, Antineoplastic↗

PubMedDate not supplied

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT).

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.

Binding Sites↗

PubMed2002

Angiogenesis stimulated by PDGF-CC, a novel member in the PDGF family, involves activation of PDGFR-alphaalpha and -alphabeta receptors.

A newly discovered PDGF isoform, PDGF-CC, is expressed in actively angiogenic tissues such as placenta, some embryonic tissues, and tumors. We test the possibility that PDGF-CC promotes angiogenesis in vivo. The core domain (mature form) of human PDGF-CC is sufficiently potent to stimulate neovascularization in the mouse cornea. The corneal angiogenic response induced by PDGF-CC is robust although the area of neovascularization is smaller than those of FGF-2- and VEGF-stimulated angiogenesis. Similarly, PDGF-BB and PDGF-AB induce angiogenic responses virtually indistinguishable from PDGF-CC-stimulated vessels. In contrast, PDGF-AA displays only a weak angiogenic response in the mouse cornea. Although there was no significant difference in incorporation of mural cells to the newly formed blood vessels induced by PDGF-BB and -CC, the percentage of mural cell positive vessels induced by PDGF-AA was greater than those induced by FGF-2, PDGF-BB, and PDGF-CC. In the developing chick embryo, PDGF-CC induced branch sprouts from established blood vessels. In PDGF receptor-transfected endothelial cells, PDGF-CC activated the PDGF receptor alpha subunit (PDGFR-alpha). PDGF-CC, but not PDGF-AA, was able to activate PDGFR-beta receptor in endothelial cells that coexpress both alpha and beta forms of receptors. Thus, the PDGF-CC-mediated angiogenic response is most likely transduced by PDGF-alphaalpha and -alphabeta receptors. These data demonstrate that the PDGF family is a complex and important group of proangiogenic factors.

Allantois↗

PubMedDate not supplied

Serum CC-10 in inflammatory lung diseases.

BACKGROUND: Although Clara cell secretory protein (CC-10) has been ascribed an anti-inflammatory role in lung diseases, its precise role remains unclear. OBJECTIVE: To further our understanding of the role of CC-10 in inflammatory lung diseases, CC-10 protein levels were measured. METHODS: Sera or bronchoalveolar lavage (BAL) fluids were collected from patients with different inflammatory lung diseases including bronchial asthma, chronic obstructive lung disease (COPD), sarcoidosis, idiopathic interstitial pneumonia (IIP), chronic eosinophilic pneumonia (CEP), pneumonia and lung cancer. Serum CC-10 concentrations were measured by enzyme-linked immunosorbent assay using urinary protein-1 antibody. Then, the relationships between CC-10 concentrations and lung diseases were investigated. Immunohistochemistry was performed using lung biopsy samples. RESULTS: Increased serum CC-10 levels were recognized in IIP patients, while CC-10 levels were decreased in bronchial asthma patients and CEP patients. Immunohistochemistry revealed an aberrant expression in areas of fibrosis in IIP patients. Serum CC-10 concentrations were not associated with severity among IIP, COPD, and sarcoidosis. In contrast, serum CC-10 concentrations were correlated with FEV(1)/FVC in bronchial asthma patients. CONCLUSIONS: Although the number of patients was quite limited, these data provide new insights into the role of CC-10 in lung diseases, and the possibility that the CC-10 concentration in serum could be a new marker indicating the severity of bronchial asthma.

Adult↗

PubMed1982

CC-1065 (NSC 298223), a novel antitumor agent that interacts strongly with double-stranded DNA.

CC-1065, a novel antibiotic produced by Streptomyces zelensis, was active against several experimental tumors in vivo and a broad spectrum of human tumor cells in vitro. This report describes its biological and biochemical effects of L1210 leukemia cells. CC-1065 is one of the most cytotoxic agents known. The concentrations required for a 50 and 90% inhibition of cell growth are 0.02 and 0.05 ng/ml, respectively. It is about 400 times more cytotoxic than was Adriamycin. The action of CC-1065 is rapid and is dose and time dependent. CC-1065 inhibits DNA synthesis much more than it inhibits RNA and protein synthesis. The concentrations required for a 50% inhibition of DNA synthesis and RNA synthesis are 4 to 6 and 45 to 60 ng/ml, respectively. Although the drug inhibition of DNA synthesis cannot completely account for its cytotoxic effects on L1210 cells, these results, along with those generated by other investigators, suggest that the inhibition of DNA synthesis represents a major mode of action of CC-1065. CC-1065 inhibited both thymidine kinase and DNA polymerase alpha activities, but the effect on highly purified DNA polymerase alpha was more pronounced. At 1 microgram/ml, CC-1065 inhibited more than 70% of the enzyme activity. In order to elucidate the mechanism of inhibition of DNA polymerase alpha, the interaction between CC-1065 and DNA was investigated. The studies with thermal melting of DNA and difference circular dichroism measurement indicate that CC-1065 is one of the strongest DNA-binding agents. It induced an increase in thermal melting temperature of calf thymus DNA by at least 31 degrees. The circular dichroism studies also reveal that CC-1065 binds only to double-stranded DNA but not to heat-denatured DNA or yeast RNA. These observations were supported by those obtained with two other experimental approaches. CC-1065 also appeared to interact with proteins, but the interaction was weak and reversible.

Animals↗

PubMed1982

Mechanism of interaction of CC-1065 (NSC 298223) with DNA.

CC-1065 (NSC 298223), a potent new antitumor antibiotic produced by Streptomyces zelensis, interacts strongly with double-stranded DNA and appears to exert its cytotoxic effects through disruption of DNA synthesis. We undertook this study to elucidate the sites and mechanisms of CC-1065 interaction with DNA. The binding of CC-1065 to synthetic and native DNA was examined by differential circular dichroism or by Sephadex chromatography with photometric detection. The binding of CC-1065 with calf thymus DNA was rapid, being complete within 2 hr, and saturated at 1 drug per 7 to 11 base pairs. The interaction of CC-1065 with synthetic DNA polymers indicated a specificity for adenine- and thymine-rich sites. Agarose gel electrophoresis of CC-1065-treated supercoiled DNA showed that CC-1065 did not intercalate. Site exclusion studies using substitutions in the DNA grooves showed CC-1065 to bind primarily in the minor groove. CC-1065 did not cause DNA breaks; it inhibited susceptibility of DNA to nuclease S1 digestion. It raised the thermal melting temperature of DNA, and it inhibited the ethidium-induced unwinding of DNA. Thus, in contrast to many antitumor agents, CC-1065 stabilized the DNA helix. DNA helix overstabilization may be relevant to the mechanism of action of CC-1065.

Animals↗

PubMed2005

Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish (Ictalurus punctatus).

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. CC chemokines constitute the largest subfamily of chemokines, with 28 CC chemokines identified from mammalian species. However, the status of CC chemokines in teleosts is yet to be determined. We previously identified 26 catfish CC chemokine cDNAs from catfish. In this study, we isolated and sequenced 23 channel catfish CC chemokine genes amounting to a total of over 56 kb of genomic sequences. Genomic organization of the 23 CC chemokine genes was determined by comparing the generated genomic sequences with the previously identified cDNA sequences. Microsatellites were identified from 16 catfish CC chemokine genes allowing them to be utilized for genome mapping. Structural analysis indicated conservation of genomic organization of CC chemokine genes, which may facilitate the establishment of orthologies. Expression of all known catfish CC chemokine transcripts was assessed in nine important tissues. Of the 26 catfish CC chemokine genes, 14 were universally expressed, six were widely expressed in many tissues, while six were highly tissue-specific.

Amino Acid Sequence↗

PubMed2000

[Cp2V] migration along an octatetrayne chain: from the monometallic complex (Cp2V(2-4eta-tBuC triple bond C2-C triple bond CC triple bond CtBu)] to the dimetallic complex.

The oxidative addition of one equivalent of [Cp2V] (4) to the tetrayne ligand tBuC triple bond CC triple bond CC triple bond CC triple bond CtBu (5) gives the monometallic complex [Cp2V(3-4eta-tBuC triple bond C-C2-C triple bond CC triple bond CtBu)] (7). Compound 7 reacts further with a second equivalent of [Cp2V] to give the dimetallic complex [(Cp2V)2(1-2eta:7-8eta-tBuC2-C triple bond CC triple bond C-C2tBu)] (8), which involves a shift of the first coordinated [Cp2V] unit from the internal C3-C4 to the external C1-C2 positions on the alkynyl ligand. Compound 8 is also directly obtained by the addition of two equivalents of [Cp2V] to 5. Reversibly, reaction of 8 with 5 leads to 7. This exchange reaction between 7 and 8 by adding successively 5 and 4 has been monitored by EPR spectroscopy. By contrast, the oxidative addition of one or two equivalents of [Cp2V] to the tetrayne ligand PhC triple bond CC triple bond CC triple bond CC triple bond CPh (6) gives the homodimetallic complex [(Cp2V)2(1-2eta:7-8eta-PhC2-CC triple bond CC triple bond C-C2-Ph)] (9). Both monometallic and dimetallic complexes 7, 8, and 9 have been characterized by X-ray diffraction. Magnetic moment measurements for 8 and 9 from 300 to 4 K indicated a weak antiferromagnetic J exchange coupling of -12.5 and -4.1 cm(-1), respectively.

Journal Article↗

PubMed1994

CC-1065 bonding to intracellular and purified SV40 DNA: site specificity and functional effects.

CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Construction and characterization of a site-directed CC-1065-N3-adenine adduct within a 117 base pair DNA restriction fragment.

The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5'-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed2006

The synthetic compound CC-5079 is a potent inhibitor of tubulin polymerization and tumor necrosis factor-alpha production with antitumor activity.

We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC(50) value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G(2)-M phase, increased phosphorylation of G(2)-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [(3)H]colchicine for binding to tubulin; however, it does not compete with [(3)H]paclitaxel (Taxol) or [(3)H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-alpha (TNF-alpha) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC(50), 270 nmol/L). This inhibitory effect on TNF-alpha production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy.

Animals↗

PubMed2005

Catfish CC chemokines: genomic clustering, duplications, and expression after bacterial infection with Edwardsiella ictaluri.

Chemokines are a family of structurally related chemotactic cytokines that regulate the migration of leukocytes, under both physiological and inflammatory conditions. CC chemokines represent the largest subfamily of chemokines with 28 genes in mammals. Sequence conservation of chemokines between teleost fish and higher vertebrates is low and duplication and divergence may have occurred at a significantly faster rate than in other genes. One feature of CC chemokine genes known to be conserved is genomic clustering. CC chemokines are highly clustered within the genomes of human, mouse, and chicken. To exploit knowledge from comparative genome analysis between catfish and higher vertebrates, here we mapped to bacterial artificial chromosome (BAC) clones 26 previously identified catfish (Ictalurus sp.) chemokine cDNAs. Through a combination of hybridization and fluorescent fingerprinting, 18 fingerprinted contigs were assembled from BACs containing catfish CC chemokine genes. The catfish CC chemokine genes were found to be not only highly clustered in the catfish genome, but also extensively duplicated at various levels. Comparisons of the syntenic relationships of CC chemokines may help to explain the modes of duplication and divergence that resulted in the present repertoire of vertebrate CC chemokines. Here we have also analyzed the expression of the transcripts of the 26 catfish CC chemokines in head kidney and spleen in response to bacterial infection of Edwardsiella ictaluri, an economically devastating catfish pathogen. Such information should pinpoint research efforts on the CC chemokines most likely involved in inflammatory responses.

Animals↗