The Lysyl Oxidase Inhibitor, β-Aminopropionitrile, Diminishes the Metastatic Colonization Potential of Circulating Breast Cancer Cells

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (μCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention

Findings to the flora of Russia and adjacent countries: New national and regional vascular plant records, 4

With this paper we continue a new annual series, the main purpose of which is to make significant floristic findings from Russia and neighboring countries more visible in Russia and abroad. In total, this paper presents new records for 48 vascular plant species from 6 Eurasian countries, obtained during field explorations, as well as during taxonomic revisions of herbarium materials. For the first time, a new locality of Leontopodium leiolepis is recorded for Russia, Rheum uzengukuushi for China, Rorippa prolifera for Lithuania, Lappula marginata for Kyrgyzstan and Tajikistan, Anthriscus caucalis, Chenopodium ficifolium, Euphorbia prostrata for Uzbekistan, Adonis × hybrida, Potamogeton × franconicus, Solidago × niederederi for the Asian part of Russia, Echinochloa esculenta, Poa jamalinensis, Puccinellia poecilantha for Siberia, Potentilla intermedia for the Caucasus, Rhynchospora alba for the Russian part of Altai, Poa sphondylodes, Veronica beccabunga for Eastern Siberia, Asclepias syriaca for the Republic of Altai, Chimaphila umbellata, Orobanche korshinskyi, Veronica scutellata for the Republic of Buryatia, Cirsium alatum, Thalictrum simplex for the Republic of Crimea, Thymus rariflorus, Th. terekensis for the Republic of Ingushetia, Berberis thunbergii, Crataegus maximowiczii, Prunus serotina for the Republic of Mordovia, Oenothera villosa for the Republic of Tatarstan, Astragalus sulcatus, Galium mollugo for the Republic of Tyva, Phragmites altissimus for the Chelyabinsk Region, Senecio dubitabilis for the Magadan Region, Asclepias syriaca, Galatella villosa, Potentilla recta for the Novosibirsk Region, Dodartia orientalis for the Omsk Region, Viola hultenii for the Sakhalin Region, Phragmites tzvelevii for the Samara Region and the Middle Volga, Jacobaea ferganensis for the Samara Region, Carex media, Impatiens parviflora for the Tyumen Region. There are some more findings which are not new for the region but they contribute significantly to the understanding of species distribution

Early vertebrate evolution of the TATA-binding protein, TBP

TBP functions in transcription initiation in all eukaryotes and in Archaebacteria. Although the 181-amino acid (aa) carboxyl (C-) terminal core of the protein is highly conserved, TBP proteins from different phyla exhibit diverse sequences in their amino (N-) terminal region. In mice, the TBP N-terminus plays a role in protecting the placenta from maternal rejection; however the presence of similar TBP N-termini in nontherian tetrapods suggests that this domain also has more primitive functions. To gain insights into the pretherian functions of the N-terminus, we investigated its phylogenetic distribution. TBP cDNAs were isolated from representative nontetrapod jawed vertebrates (zebrafish and shark), from more primitive jawless vertebrates (lamprey and hagfish), and from a prevertebrate cephalochordate (amphioxus). Results showed that the tetrapod N-terminus likely arose coincident with the earliest vertebrates. The primary structures of vertebrate N-termini indicates that, historically, this domain has undergone events involving intragenic duplication and modification of short oligopeptide-encoding DNA sequences, which might have provided a mechanism of de novo evolution of this polypeptide

Fundamental cellular processes do not require vertebrate-specific sequences within the TATA-binding protein

The 180-amino acid core of the TATA-binding protein (TBPcore) is conserved from Archae bacteria to man. Vertebrate TBPs contain, in addition, a large and highly conserved N-terminal region that is not found in other phyla. We have generated a line of mice in which the tbp allele is replaced with a version, tbp(Delta N), which lacks 111 of 135 N-terminal amino acid residues. Most tbp(Delta N/Delta N) fetuses die in midgestation. To test whether a disruption of general cellular processes contributed to this fetal loss, primary fibroblast cultures were established from +/+, Delta N/+, and Delta N/Delta N fetuses. The cultures exhibited no genotype-dependent differences in proliferation or in expression of the proliferative markers dihydrofolate reductase (DHFR) mRNA (S phase-specific) and cdc25B mRNA (G(2)-specific). The mutation had no effect on transcription initiation site fidelity by either RNA polymerase II (pol II) or pol III. Moreover, the mutation did not cause differences in levels of U6 RNA, a pol III-dependent component of the splicing machinery, in mRNA splicing efficiency, in expression of housekeeping genes from either TATA-containing or TATA-less promoters, or in global gene expression. Our results indicated that general eukaryotic cell functions are unaffected by deletion of these vertebrate-specific sequences from TBP. Thus, all activities of this polypeptide domain must either be compensated for by redundant activities or be restricted to situations that are not represented by primary fibroblasts

Reproductive and neurological Quakingviable phenotypes in a severe combined immune deficient mouse background

The quaking viable ( qk v ) mutation, a spontaneous deletion of a multigenic region encompassing roughly 1 Mb at 5.9 cM on the proximal end of mouse chromosome 17, causes severe trembling in all homozygous animals and infertility in all homozygous males. Physiologically, quaking mice exhibit dysmyelination and postmeiotic spermatogenic arrest. Molecular defects in Qk v mice occur in the affected tissues, indicating the primary causes of these pathologies are cell autonomous. However, because both the reproductive and neurological defects are in immune-privileged sites and because some similar pathologies at both sites have been shown to be immune mediated, we tested whether the immune system participates secondarily in manifestation of Qk v phenotypes. The qk v mutation was bred into a severe combined immune-deficient mouse line (SCID; devoid of mature B and T cells) and penetrance of the neurological and the male sterile phenotypes was measured. Results showed that neither defect was ameliorated in the immune-deficient background. We conclude that the Qk v pathologies do not likely involve a B- or T-cell-dependent response against these immune-privileged sites

Removing the Vertebrate-Specific TBP N Terminus Disrupts Placental β2m-Dependent Interactions with the Maternal Immune System

Mammalian TBP consists of a 180 amino acid core that is common to all eukaryotes, fused to a vertebrate-specific N-terminal domain. We generated mice having a modified tbp allele, tbp ΔN , that produces a version of TBP lacking 111 of the 135 vertebrate-specific amino acids. Most tbp ΔN/ΔN fetuses (>90%) died in midgestation from an apparent defect in the placenta. tbp ΔN/ΔN fetuses could be rescued by supplying them with a wild-type tetraploid placenta. Mutants also could be rescued by rearing them in immunocompromised mothers. In immune-competent mothers, survival of tbp ΔN/ΔN fetuses increased when fetal/placental β2m expression was genetically disrupted. These results suggest that the TBP N terminus functions in transcriptional regulation of a placental β2m-dependent process that favors maternal immunotolerance of pregnancy

Evaluation of knee osteolysis by μCT.

<p>Mice undergoing BAPN treatment and their controls were sacrificed at day 21 after intracardiac injection of MDA-MB-231-Luc2 cells, and their left hind limbs were examined by μCT. Hind limbs from age-matched non-tumor cell-injected mice were used as controls. Anterior, posterior, and medial μCT images of representative knee joints from the different groups are shown (A). Bone volumes were determined from measurements of three hundred 10 µm slices taken just distal to the proximal tibial growth plate and graphed as mean bone volume±SEM (B) (n = 8 for ‘no IC’, that is, control mice that were not injected with tumor cells; n = 9 for ‘no BAPN’; n = 10 for ‘BAPN d 7’; and n = 5 for ‘BAPN d −1’ groups). Asterisk indicates a statistical significance of P<0.05.</p

<i>In vitro</i> evaluation of MDA-MB-231-Luc2 cells.

<p>(A) Cells were plated in a 96-well plate in quadruplicate, ranging from 10,000 to 2,000 cell/well. Wells with media only (no cells) were included as control. <i>D</i>-luciferin substrate was added to each well and the plate was imaged to estimate the bioluminescence of MDA-MB-231-Luc2 cells. (B) Correlation between bioluminescence and cell number per well was plotted as mean photons/s/well±SEM. (C) Fluorescence microscopy, showing that MDA-MB-231-Luc2 cells expressed EGFP. (D) 1×10³ MDA-MB-231 parental and MDA-MB-231-Luc2 cells were seeded into quadruplicate wells of 96-well plates and their proliferation rates determined by MTT assay. Data was plotted as mean relative growth±SEM.</p

Growth of MDA-MB-231-Luc2 metastasis and the effects of BAPN treatment.

<p>Bioluminescence imaging was performed 2 times per week starting on day 7 after intracardiac injection of tumor cells. Representative dorsal images of control and BAPN-treated mice are shown from 7 to 21 d post-injection (A). Images for days 7–10, and days 14 to 21 are shown with different sensitivity color scale bars, reflecting the rapid growth rate of metastases. Scales are in photons/s/cm². Whole body luminescence, a measure of tumor burden, for the different groups is shown as a function of time (B); note the need for a log scale owing to the rapid growth of the tumors. BAPN treatment was initiated at the indicated times and then continued daily thereafter until day 21. (C) Whole body bioluminescence at day 7 post-cell injection was quantified as mean photons/s±SEM. Whole body tumor bioluminescence at day 21 (D) was quantified as mean photons/s±SEM. For both (B) and (C), n = 10 for each of ‘no BAPN’ and for ‘BAPN d 7’ groups, and n = 5 for ‘BAPN d −1’ and for ‘BAPN d 0’ groups. Asterisks indicate statistical significance (^* P<0.05;^** P<0.01; ^*** P<0.001).</p