Integrative analysis of gene expression patterns predicts specific modulations of defined cell functions by estrogen and tamoxifen in MCF7 breast cancer cells.

To explore the mechanisms whereby estrogen and antiestrogen (tamoxifen (TAM)) can regulate breast cancer cell growth, we investigated gene expression changes in MCF7 cells treated with 17beta-estradiol (E2) and/or with 4-OH-TAM. The patterns of differential expression were determined by the ValiGen Gene IDentification (VGID) process, a subtractive hybridization approach combined with microarray validation screening. Their possible biologic consequences were evaluated by integrative data analysis. Over 1000 cDNA inserts were isolated and subsequently cloned, sequenced and analyzed against nucleotide and protein databases (NT/NR/EST) with BLAST software. We revealed that E2 induced differential expression of 279 known and 28 unknown sequences, whereas TAM affected the expression of 286 known and 14 unknown sequences. Integrative data analysis singled out a set of 32 differentially expressed genes apparently involved in broad cellular mechanisms. The presence of E2 modulated the expression patterns of 23 genes involved in anchors and junction remodeling; extracellular matrix (ECM) degradation; cell cycle progression, including G1/S check point and S-phase regulation; and synthesis of genotoxic metabolites. In tumor cells, these four mechanisms are associated with the acquisition of a motile and invasive phenotype. TAM partly reversed the E2-induced differential expression patterns and consequently restored most of the biologic functions deregulated by E2, except the mechanisms associated with cell cycle progression. Furthermore, we found that TAM affects the expression of nine additional genes associated with cytoskeletal remodeling, DNA repair, active estrogen receptor formation and growth factor synthesis, and mitogenic pathways. These modulatory effects of E2 and TAM upon the gene expression patterns identified here could explain some of the mechanisms associated with the acquisition of a more aggressive phenotype by breast cancer cells, such as E2-independent growth and TAM resistance.

Inteins of Thermococcus fumicolans DNA polymerase are endonucleases with distinct enzymatic behaviors.

The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes. Both inteins have been produced in Escherichia coli and purified either as naturally spliced products from the expression of the complete DNA polymerase gene or directly from the cloned inteins genes. Both recombinant inteins exhibit endonuclease activity, with an optimal temperature of 70 degrees C. The Tfu pol-1 intein, which belongs to the Psp KOD pol-1 allelic family, recognizes and cleaves a minimal sequence of 16 base pairs (bp) on supercoiled DNA with either Mn(2+) or Mg(2+) as cofactor. It cleaves linear DNA only with Mn(2+) and requires a 19-bp minimal recognition sequence. The Tfu pol-2 intein, which belongs to the Tli pol-2 allelic family, is a highly active homing endonuclease using Mg(2+) as cofactor. Its minimal recognition and cleavage site is 21 bp long either on linear or circular DNA substrates. Its endonuclease activity is strongly inhibited by the 3' digestion product, which remains bound to the enzyme after the cleavage reaction. According to current nomenclature, these endonucleases were named PI-TfuI and PI-TfuII. These two inteins thus exhibit different requirements for metal cofactor and substrate topology as well as different mechanism of action.

Cytometric detection of mycobacterial surface antigens: exposure of mannosyl epitopes and of the arabinan segment of arabinomannans.

The physical arrangement of cell envelope components leads to the exposure of selected structural motifs which in turn may influence host-parasite interactions. To gain insight into the exposed epitopes, the present study describes a flow cytometric method designed to probe defined molecules on dispersed mycobacteria. The hydrophobic fluorophore N-hexadecanoyl aminofluorescein inserted in the mycobacterial cell envelope permitted focusing of fluorescence-activated cell sorter analysis on cells that were further labeled with defined monoclonal antibodies and fluorochrome-coupled streptavidin. The use of antibodies directed against the lipooligosaccharide of Mycobacterium tuberculosis demonstrated the specific detection of the antigen on the cell surface of a Canetti-like strain of M. tuberculosis, and not on those of mycobacterial strains that were devoid of the glycolipid. Thus, the method was applied to investigate the relative amounts of surface-exposed mannosylated compounds and D-arabinan-containing substances of different strains of the tubercle bacillus and a strain of the rapidly growing nonpathogenic species Mycobacterium smegmatis. Both M. tuberculosis and M. smegmatis are endowed with mannosyl and arabinan epitopes on their surfaces, although there are many differences in terms of exposed mannosyl epitopes between the various strains of the tubercle bacillus examined. These differences are correlated with the amounts of terminal mannosyl residues that cap the surface-exposed arabinomannans (A. Ortalo-Magné, A. B. Andersen, and M. Daffé, Microbiology 142:927-935, 1996) but not with the degrees of virulence of the strains. This novel approach could provide new insights into the distribution of defined surface-exposed antigens and thereby into the architecture of the cell envelopes.