Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression

International audienceBackground: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. Methodology/Principal Findings: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G 1 phase and hTS is localized in the nuclei during S and G 2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies. Cop. 2012 Ligabue et al

Forebrain CRF₁ modulates early-life stress-programmed cognitive deficits

Childhood traumatic events hamper the development of the hippocampus and impair declarative memory in susceptible individuals. Persistent elevations of hippocampal corticotropin-releasing factor (CRF), acting through CRF receptor 1 (CRF1), in experimental models of early-life stress have suggested a role for this endogenous stress hormone in the resulting structural modifications and cognitive dysfunction. However, direct testing of this possibility has been difficult. In the current study, we subjected conditional forebrain CRF1 knock-out (CRF1-CKO) mice to an impoverished postnatal environment and examined the role of forebrain CRF1 in the long-lasting effects of early-life stress on learning and memory. Early-life stress impaired spatial learning and memory in wild-type mice, and postnatal forebrain CRF overexpression reproduced these deleterious effects. Cognitive deficits in stressed wild-type mice were associated with disrupted long-term potentiation (LTP) and a reduced number of dendritic spines in area CA3 but not in CA1. Forebrain CRF1 deficiency restored cognitive function, LTP and spine density in area CA3, and augmented CA1 LTP and spine density in stressed mice. In addition, early-life stress differentially regulated the amount of hippocampal excitatory and inhibitory synapses in wild-type and CRF1-CKO mice, accompanied by alterations in the neurexin-neuroligin complex. These data suggest that the functional, structural and molecular changes evoked by early-life stress are at least partly dependent on persistent forebrain CRF1 signaling, providing a molecular target for the prevention of cognitive deficits in adults with a history of early-life adversity

Non-existence of a dilaton gravity action for the exact string black hole

We prove that no local diffeomorphism invariant two-dimensional theory of the metric and the dilaton without higher derivatives can describe the exact string black hole solution found a decade ago by Dijkgraaf, Verlinde and Verlinde. One of the key points in this proof is the concept of dilaton-shift invariance. We present and solve (classically) all dilaton-shift invariant theories of two-dimensional dilaton gravity. Two such models, resembling the exact string black hole and generalizing the CGHS model, are discussed explicitly.Comment: 24 pages, 3 eps-figures, revised version (more references, clarified some of the discussion

Magnetic measurement methods to probe nanoparticle–matrix interactions

Magnetic nanoparticles (MNPs) are key elements in several biomedical applications, e.g., in cancer therapy. Here, the MNPs are remotely manipulated by magnetic fields from outside the body to deliver drugs or generate heat in tumor tissue. The efficiency and success of these approaches strongly depend on the spatial distribution and quantity of MNPs inside a body and interactions of the particles with the biological matrix. These include dynamic processes of the MNPs in the organism such as binding kinetics, cellular uptake, passage through cell barriers, heat induction and flow. While magnetic measurement methods have been applied so far to resolve the location and quantity of MNPs for therapy monitoring, these methods can be advanced to additionally access these particle–matrix interactions. By this, the MNPs can further be utilized as probes for the physical properties of their molecular environment. In this review, we first investigate the impact of nanoparticle–matrix interactions on magnetic measurements in selected experiments. With these results, we then advanced the imaging modalities magnetorelaxometry imaging and magnetic microsphere tracking to spatially resolve particle–matrix interactions

Magnetic measurement methods to probe nanoparticle–matrix interactions

Magnetic nanoparticles (MNPs) are key elements in several biomedical applications, e.g., in cancer therapy. Here, the MNPs are remotely manipulated by magnetic fields from outside the body to deliver drugs or generate heat in tumor tissue. The efficiency and success of these approaches strongly depend on the spatial distribution and quantity of MNPs inside a body and interactions of the particles with the biological matrix. These include dynamic processes of the MNPs in the organism such as binding kinetics, cellular uptake, passage through cell barriers, heat induction and flow. While magnetic measurement methods have been applied so far to resolve the location and quantity of MNPs for therapy monitoring, these methods can be advanced to additionally access these particle–matrix interactions. By this, the MNPs can further be utilized as probes for the physical properties of their molecular environment. In this review, we first investigate the impact of nanoparticle–matrix interactions on magnetic measurements in selected experiments. With these results, we then advanced the imaging modalities magnetorelaxometry imaging and magnetic microsphere tracking to spatially resolve particle–matrix interactions

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed the mutant strain BL03 that was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active clones in the thermophilic bacterium than in the mesophilic E. coli. From all clones functionally screened in E. coli, only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance

Revisiting caveolin trafficking: the end of the caveosome

In this issue, a study by Hayer et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003086) provides insights into the trafficking of caveolins, the major membrane proteins of caveolae. As well as providing evidence for ubiquitin-mediated endosomal sorting and degradation of caveolin in multivesicular bodies (MVBs), the new findings question the existence of a unique organelle proposed nine years ago, the caveosome