Conformational stabilization of FOX-DNA complex architecture to sensitize prostate cancer chemotherapy.

The forkhead box (FOX) transcription factor is a family of tumor suppressors that negatively regulates the tumorigenesis activity of prostate cancer; stabilization of FOX-DNA complex architecture has been recognized as a new and promising strategy for sensitizing cancer chemotherapy. Here, we described a systematic method that combined in silico analysis and in vitro assay to investigate the intermolecular interaction between FOX DNA-binding domain (DBD) and its cognate DNA partner. The structural and energetic information harvested from the molecular investigation were used to guide high-throughput virtual screening against a structurally diverse, nonredundant library of natural product compounds, aiming at discovery of novel small-molecule medicines that can conformationally stabilize and promote FOX-DNA recognition and interaction. The screening identified a number of theoretically promising hits, which were then examined by using fluorescence anisotropy assay to determine their binding potency for FOX DBD domain. The antitumor activity of identified high-affinity compounds was also tested at cellular level. Structural dynamics analysis found that the small-molecule stabilizers can shift the conformational equilibrium of FOX DBD to DNA-bound state, thus promoting the protein domain to bind tightly with its DNA partner.

[Comparative study of three antineoplastic alkylating agents on DNA]. / Sravnitel'noe issledovanie vliianiia trekh alkiliruiushchikh protivoopukholevykh preparatov na DNK.

The influence of three alkylating anticancer preparations phosphamide, sarcolysine, cyclophosphane on content of the 5-methylcytosine and parameters of the melting DNA of the liver healthy animals and tumor sarcoma 45 was investigated. It was shown, that among the investigated preparations cyclophosphane has stronger anticancer influence and comparatively weaker side effect on DNA liver. We came to the conclusion that it is preferable to use this preparation.

A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells.

The human cMOAT gene encodes a membrane protein involved in the ATP-dependent transport of hydrophobic compounds. To determine whether cMOAT is associated with drug sensitivity, we transfected an expression vector containing cMOAT antisense cDNA into the HepG2 human hepatic cancer cell line. We observed a reduction in cMOAT protein, as well as an enhanced level of glutathione, in the antisense transfectants. The transfectants displayed an increased sensitivity to cisplatin, vincristine, doxorubicin, and the camptothecin derivatives, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyl oxy]dione hydrochloride triethydrate and 7-ethyl-10-hydroxycamptothecin, but not to etoposide, 3-[4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosoure a, 5-fluorouracil, and mitomycin C. Results suggest that cMOAT levels are inversely correlated with those of glutathione, and that cMOAT and its related genes may be involved in the sensitivity of cells to certain anticancer agents.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 enhance the incorporation of cytosine arabinoside into the DNA of leukemic blasts and the cytotoxic effect on clonogenic cells from patients with acute myeloid leukemia.

In the present study the effects of the 48-hour administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 U/mL) or interleukin-3 (IL-3) (100 U/mL) on the proliferative activity of leukemic cells and on the intracellular metabolism and cytotoxic efficacy of a subsequent 12-hour application of cytosine arabinoside (ara-C) at doses of 0.1, 1.0, 10.0, and 100.0 mumol/L were evaluated on bone marrow cells from 17 patients with acute myeloid leukemia. After GM-CSF or IL-3, a 1.2- to 2.4-fold increase in S-phase cells was observed in nine of 14 GM-CSF and seven of 11 IL-3 cases. 3H-Cytosine arabinoside incorporation into the DNA was enhanced 1.33- to 18.3-fold over respective controls in 14 of 17 patients. While in control specimens are ara-C dose-dependent increase in 3H-ara-C uptake was accompanied by a corresponding rise in intracellular ara-C-5' triphosphate (ara-CTP) levels, ara-CTP concentrations were not increased after GM-CSF or IL-3 exposure, resulting in a higher ara-C to ara-CTP ratio over controls. This finding may be explained by a stimulatory effect of GM-CSF and IL-3 on ara-C phosphorylating enzymes and a more rapid incorporation of ara-CTP into the DNA of leukemic blasts. These effects translated into a 2.2- to 229.0-fold increase in the cytotoxic activity of ara-C against clonogenic leukemic cells after GM-CSF or IL-3 pretreatment. Hence, GM-CSF and IL-3 enhance the intracellular metabolism of ara-C and its incorporation into the DNA of leukemic cells leading to a higher antileukemic activity of ara-C on clonogenic leukemic cells (CFU-L).

Regulation of human lymphocyte proliferation by a tumour cell-derived DNA fraction.

A suppressor activity was isolated and partially purified from culture supernatant of human myeloid leukaemic cell line HL-60 by ion-exchange chromatography, HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This activity was capable of suppressing proliferation of mitogen-stimulated normal human lymphocytes in a dose-dependent manner. The suppressor activity was identified as DNA by its typical absorbance at 260 nm, and by its susceptibility to DNase digestion. The DNA with suppressive activity was found to be double-stranded with the molecular weight range between 0.6 and 1.5 kb. This DNA material did not have cytotoxic activity and did not alter interleukin-2 receptor expression. Cell cycle analysis has suggested that DNA molecules could interact with lymphocytes to arrest the stimulated lymphocytes in the G1 phase, apparently preventing the cells from entering into the proliferating S phase.

[The DNA-mediated transformation of mouse fibroblasts after radiation damage to the cellular chromatin]. / DNK-oposredovannaia transformatsiia fibroblastov myshei posle radiatsionnogo povrezhdeniia kletochnogo khromatina.

In experiments with NIH3T3 mouse fibroblast the authors showed a dose-dependent (gamma- or UV-radiation) increase in the immortalized fibroblast transformation by purified DNA preparations from Ehrlich ascites tumour cells. The transformant yield was the highest when the transfection started within the first few minutes after irradiation, when radiation lesions occurred in the genome and the system of enzymic DNA repair was activated. The proteolytic activity inhibition by treating the exposed cells with phenylmethanesultonyl fluorine reduced the radiation-induced transformation.

Transforming activity of DNA from rat liver tumors induced by the carcinogen methyl(acetoxymethyl)nitrosamine.

Altered c-Ha-ras genes have been frequently detected in the DNA of spontaneous or chemically induced mouse liver tumors. To determine if ras gene mutation is a frequent event during liver carcinogenesis in rats, we examined the transforming activity of DNA from liver tumors that developed in rats injected with methyl(acetoxymethyl)nitrosamine (DMN-OAc) after a partial hepatectomy. Three weeks after the injection of DMN-OAc, rats were fed a diet containing phenobarbital. This carcinogen acts only on replicating liver cells. Six of eight tumor DNAs induced the transformation of NIH 3T3 cells. The transforming activity was stable upon a second round of transfection, and the transformants were tumorigenic in nude mice. Southern blot analysis of transformant DNAs showed that the transforming activity was not due to the acquisition of a ras (Ha, Ki, or N), neu, myc, A-raf, v-raf, erbA, or erbB gene of rat origin. Several transformants' restriction enzyme sensitivity was analyzed, and their activity indicated that similar transforming sequences were present in at least two tumors and that one tumor contained two different transforming sequences. These results suggest that during hepatocarcinogenesis induced in rats by DMN-OAc, alterations in the ras gene family occur infrequently or not at all and that several different genes (which are not homologous to common oncogenes) become activated and are capable of transforming NIH 3T3 cells.

[Transforming activity of the DNA from human hepatoma cells. The transcription of integrated viral sequences and their cloning]. / Transformiruiushchaia aktivnost' DNK kletok gepatomy cheloveka. Transkriptsiia integrirovannykh virusnykh posledovatel'nostei i ikh klonirovanie.

A monolayer culture of mouse fibroblasts, line NIH/3T3, was transformed by DNA from human hepatoma cell line PLC/PRF/5. The resulting lines of transformed cells differ in their growth characteristics and are capable of producing tumors in nude mice. DNA of these cell lines was found to contain integrated sequences of human DNA and hepatitis B virus DNA. Analysis of transcription showed the amount of virus-specific RNA in the original cell line PLC/PRF/5 to be 20 times as large as in one of the transformed lines. Fragments of DNA of the transformed cells containing virus sequences were cloned, and 400,000 clones were obtained. A clone was identified the insert of which contains a portion of viral genome.

Research in the field of nucleic acids performed in the "Stefan S. Nicolau" Institute of Virology.

A review is made of the research in the field of nucleic acids performed in the "Stefan S. Nicolau" Institute of Virology. The results obtained as regards the infectivity of viral nucleic acids, the oncogenic capacity of nucleic acids extracted from tumors, the isolation, characterization, physicochemical and biological activity of viral and cellular nucleic acids, as well as some achievements in recombinant DNA technology, are briefly presented.

Enhancement of the biological activities of deoxyribonucleic acid extracted from canine venereal tumours.

Deoxyribonucleic acid (DNA) extracted from canine venereal tumours was capable of changing normal cells to morphologically abnormal cells in an in vitro system of L-fibroblasts. The observed effects were proportional to the concentration of the nucleic acid extract used; the higher the concentration, the more pronounced the effect. Inactivation of this effect by deoxyribonuclease indicated that the active nucleic acid moiety was DNA. Purified RNA did not appear to contribute to the observed activity. The activity was enhanced by diethyl-amino ethyl-dextran (DEAE-D) and dimethyl sulphoxide (DMSO) and the data showed that 20% DMSO was superior to 300 g/ml DEAE-D as an enhancing agent.

Isolation from murine myeloma and leukemia cells of a selective inhibitor of DNA polymerases.

An inhibitor of two ribopolymer-transcribing DNA polymerases can be isolated from MOPC-21 myeloma tumors, from the sera of mice bearing these tumors, or from MCDV-12 ascitic leukemia. The peritoneal fluid of mice bearing the latter neoplasm is also a source of the inhibitor. The inhibitor is a small DNA molecule which displays inhibitory activity in both double- and single-stranded form.

Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.

An ATPase depending on the presence of single-stranded DNA from mouse myeloma.

An ATPase was purified from mouse myeloma MOPC 70E the activity of which depends on the presence of single-stranded DNA and divalent cations such as Mg2+, Mn2+, Ca2+, Ni2+ or Fe2+. The enzyme splits both ribonucleoside and deoxyribonucleoside triphosphates but preferentially ATP and dATP yielding nucleoside diphosphates and inorganic phosphate. The enzyme has an absolute requirement for single-stranded DNA. Alternating double-stranded polydeoxynucleotides are only slight effective, and native double-stranded DNA, single-stranded and double-stranded RNAs as well as DNA - RNA hybrids are ineffective in stimulating the ATPase. The enzyme has further characterized by sedimentation in a sucrose density gradient (s20, w = 5.5 S) and by isoelectric focussing in an ampholine pH gradient (pI = 6.5).