The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis.

Nanog is a transcription factor required for maintaining the pluripotency of embryonic stem cells, and is not expressed in most normal adult tissues. However, recent studies have indicated that Nanog is overexpressed in many types of human cancers, including breast cancer. To elucidate the physiological roles of Nanog in tumorigenesis, we developed an inducible Nanog transgenic mouse model, in which the expression of Nanog in adult tissues can be induced via LoxP/Cre-mediated deletion. Our findings indicate that overexpression of Nanog in the mammary gland is not sufficient to induce mammary tumor. However, when coexpressed with Wnt-1 in the mouse mammary gland, it promotes mammary tumorigenesis and metastasis. In this context, Nanog promotes the migration and invasion of breast cancer cells. Microarray analysis has shown that the ectopic expression of Nanog deregulates the expression of numerous genes associated with tumorigenesis and metastasis, such as the PDGFRα gene. Our findings demonstrate the involvement of Nanog in breast cancer metastasis, and provide the basis for the reported correlation between Nanog expression and poor prognosis of human breast cancer patients. As Nanog is not expressed in most adult tissues, these findings identify Nanog as a potential therapeutic target in the treatment of Nanog-expressing metastatic breast cancer.

Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action.

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

Preferential binding of tumor suppressor p53 to positively or negatively supercoiled DNA involves the C-terminal domain.

The C-terminal domain of the tumor suppressor protein p53 is the site of non-specific DNA binding. Purified p53 produced from baculovirus-infected insect cells binds preferentially to supercoiled DNA, forming bands with lower electrophoretic mobility. This non-covalent binding does not change the linking number of the DNA. An anti-p53 antibody targeting the C-terminal domain partially competes with supercoiled DNA in binding to p53, while antibodies targeted to the N terminus of p53 supershift the complex bands. A synthetic peptide corresponding to amino acid residues 319-393 of human p53 also displays preferential binding to supercoiled DNA, while a mutant peptide, which is unable to form tetramers, is inactive. The center of the equilibrium distribution of topoisomers formed by relaxation with topoisomerase I is not shifted in the presence of p53 although the distribution is broadened. The preferential binding of p53 is exhibited toward both positively and negatively supercoiled DNA. These observations are consistent with a model in which p53 binds to right-handed or left-handed strand crossings.

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.

Inhibition of RNA polymerase II transcription in human cell extracts by cisplatin DNA damage.

The anticancer drug cisplatin induces a spectrum of lesions in DNA. The effect of such DNA damage on transcription by RNA polymerase II (RNA pol II) in human cell extracts was investigated at the level of initiation and elongation. RNA pol II transcription directed from the adenovirus major late promoter was inhibited following treatment of the promoter-containing template with increasing concentrations of cisplatin. Furthermore, transcription from an undamaged promoter fragment was depleted in the presence of increasing amounts of cisplatin DNA damage on an exogenous plasmid, suggesting such damage may hijack an essential factor for transcription initiation. The effect of cisplatin damage on RNA pol II elongation was investigated using site-specifically-placed cisplatin adducts. The GTG adduct was an effective block to RNA pol II elongation, inhibiting the polymerase by 80%. In contrast, RNA pol II completely bypassed the cisplatin GG intrastrand adduct. These studies suggest that the inhibition of RNA pol II transcription observed following the treatment of cells with cisplatin is likely to reflect the combined effects of DNA damage at the level of both transcription initiation and elongation.

Stereospecific differences in repair by human cell extracts of synthesized oligonucleotides containing trans-opened 7,8,9, 10-tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N2-dG adduct stereoisomers located within the human K-ras codon 12 sequence.

The potent environmental carcinogen benzo[a]pyrene (BaP), following enzymatic activation to enantiomeric pairs of bay-region 7,8-diol 9, 10-epoxides (the benzylic 7-hydroxyl group and epoxide oxygen are cis for DE-1 diastereomers and trans for DE-2 diastereomers), reacts with DNA to form covalent adducts predominately at the exocyclic amino groups of purines. Specific adducts, corresponding to the trans opening of each of the four optically active BaP DE isomers at C-10 by the N 2-amino group of dG, were synthesized as appropriately blocked phosphoramidites and were incorporated at either the first or second G of codon 12 within the G-rich sequence of human K-ras codons 11-13: GCT G1G2T GGC. The adducted oligonucleotides were incorporated into plasmids by primer extension, followed by purification of the covalently closed circular constructs. Adducts derived from either (+)- or (-)-DE-2, placed at either G1 or G2, presented strong blocks to in vitro transcription elongation by bacteriophage T3 RNA polymerase, but only moderately blocked transcription elongation by human RNA polymerase II in nuclear extracts. Adducts derived from all four DEs, placed on either G1 or G2, were used as substrates in a DNA repair synthesis assay using human whole cell extracts. Adducts derived from three of the DE stereoisomers exhibited significant amounts of repair synthesis, but the (-)-DE-2 adduct experienced no repair synthesis above that of the control. Constructs containing a pre-existing nick at the sixth phosphodiester bond 3' to either (+)-DE-2 or (-)-DE-2 adducts exhibited increased repair synthesis.

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.

Dimerization of Escherichia coli UvrA and its binding to undamaged and ultraviolet light damaged DNA.

The initial stages in the repair of damaged DNA by the Escherichia coli uvr system involve the recognition of damage by UvrA. We have examined in detail the binding of UvrA to DNA randomly damaged by ultraviolet light, undamaged DNA, and single-stranded DNA using nitrocellulose filter binding and gel mobility shift assays to arrive at the following model: UvrA dimers bind specifically to damaged DNA both in the presence and in the absence of ATP. The dimerization of UvrA is promoted by UvrA concentrations greater than 1 nM, the presence of ATP, or physiological temperatures, and the dimerization step dominates the temperature dependence of UvrA binding to DNA damaged by ultraviolet light. The apparent association constant for specific binding is dependent on the concentration of UvrA due to coupled dimerization, aggregation, and nonspecific binding reactions. At 1 nM UvrA, either with or without ATP, Kuv approximately 10(9) M-1. The binding of UvrA to undamaged DNA is 10(3)-10(4)-fold weaker than the damage-specific binding. Both the strength of damage-specific binding and the discrimination between damaged and undamaged sites are affected by the salt concentration. The kinetics of association and dissociation reactions indicate that the primary effects of ATP are on the extent of UvrA dimerization rather than on the properties of the UvrA-uvDNA complex. The complexity of the interaction of UvrA, ATP, and DNA is indicated by the opposing effects of ATP binding and hydrolysis on UvrA dimerization.

Association kinetics of site-specific protein-DNA interactions: roles of nonspecific DNA sites and of the molecular location of the specific site.

We have applied the formalism developed previously for the kinetics of domain-localized reactions [S. Mazur and M. T. Record, Jr. (1986) Biopolymers 25, 985-1008] to describe complex mechanisms of association of a protein with a specific site on a large DNA molecule also containing many nonspecific binding sites. These nonspecific sites participate in the mechanism of formation of the specific complex through competitive binding and the facilitating mechanisms of sliding and transfer. The effects of localizing the sites in a domain are represented by a simple algebraic expression, and the sequence of interactions within the domain are described by equations closely related to a conventional, homogeneous solution mechanism. We apply this formalism to examine the interplay between sliding and direct transfer in domain-localized interactions in general and in the lac repressor-lac operator interaction in particular. Experimental investigation of the effect of the molecular location of the specific site (e.g., end vs middle of the polymer chain) on the kinetics of association may allow the contributions of sliding and direct transfer to be resolved.

Dynamics of the Escherichia coli nucleotide excision repair system.

Ultraviolet light induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. At the highest level of complexity Escherichia coli (E. coli) has a uvr DNA repair system comprising the UvrA, UvrB and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding, is associated with localized topological unwinding of DNA. This protein complex can catalyze an ATP-dependent 5'----3' directed strand displacement of D-loop DNA or short single strands annealed to a single stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled "excision resynthesis" step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress induced protease which also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.

Repair of DNA-containing pyrimidine dimers.

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.