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Pitx2 expression induces cell cycle exit and p21 expression in neural stem cells
Cortical development is a complex process that involves many events including proliferation, cell cycle exit, and differentiation that need to be appropriately synchronized.. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. The G1 phase of the cell cycle is mostly associated with cell cycle arrest and cell differentiation. Cell cycle progression and exit during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2, and found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≈20-fold) increase in expression of the cell cycle inhibitor p21Cip. In addition, Pitx2 bound directly to the p21Cip promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 directly regulates p21Cip expression and induces cell cycle exit in neural progenitors.Keywords: Neocortex, p27Kip2, Chromatin immunoprecipitation, Chromatin, p21Cip1, Transcription, Neural progenitors, p57Kip2, Pitx, Telencephalo
Fatty Acids Derived from Royal Jelly Are Modulators of Estrogen Receptor Functions
Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E2), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E2 FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E2-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E2-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E2, FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E2, FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E2, induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJ's components which, although structurally entirely different from E2, mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes
Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells
AbstractThe Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells
Molecular basis of estrogen receptor antagonism
Estrogen Receptors (ERs), ERa and ERb, are responsible for mediating the
physiological effects of the steroid hormone 17b-estradiol and
structurally related compounds. ER signaling is a complex process where
the combined effects of accessory proteins, known as coregulators,
promoter characteristics and influence from other signaling systems,
determine the cell-specific transcriptional response in a given
situation. Central to the regulatory mechanism is the ability of ERs to
adopt distinct conformations in response to ligands and thereby determine
the relative affinity of coregulators. Dysregulation of ER signaling
leads to increased cell proliferation and reproductive diseases.
Synthetic anti-estrogens can antagonize inappropriate ER signaling
constituting the rationale behind their clinical use in cancer therapy.
The mechanisms that govern anti-estrogen action are poorly understood
with several of the currently applied drugs having unwanted side effects
in non-target tissues, and after prolonged treatment tumor cells become
resistant to anti-estrogen therapy. Recent findings extend the
traditional view of ER antagonists as compounds that solely block
estrogen effects to include “active antagonist receptor signaling”.
The aim of this thesis was to better understand the molecular details
involved in mediating the effects of antagonist-bound ER complexes. To
this end we have investigated coregulator interaction surfaces on ERs
using peptides that mimic coregulator interactions. A major finding of
our studies is that the ER-ligand-binding domains harbor surfaces capable
of recruiting coregulators in the presence of antagonists, which may
represent bona fide control surfaces involved in mediating antagonist
activity. We identified peptide interactions that can serve as models for
coregulator mediated subtype and antagonist specific effects (Paper I)
and a peptide interaction (Paper II) that serve as a model for
coregulator mediated agonist and antagonist action. In addition, a
peptide interaction-mechanism overlapping with a known nuclear receptor
(NR) corepressor surface was characterized with detailed structural
analysis (Paper III). Moreover, the peptide-receptor interaction system
turned out to be a very useful system for monitoring receptor
conformation after binding of uncharacterized ligands. Phytoestrogens,
such as genistein, are being extensively investigated in search of new
therapeutic agents for estroge n-related pathologies. 8-prenylnaringenin
is a potent plantderived estrogenic compound, whereas naringenin lacking
a prenyl-group is not. We therefore specifically investigated the impact
of alkyl chain length and degree of branching at C(8) of the naringenin
skeleton on estrogenic effects (Paper IV). Taken together, the in vitro
bioassays of the 10 novel naringenin derivatives, accessed by a
regioselective alkylation scheme at C(8), showed a very interesting
relationship between the nature of the alkyl substituent and the
established bioactivity. They span an activity spectrum ranging from full
agonists to partial agonists to antagonists. Our systematic approach to
investigate the impact of the nature of the substituent provides further
evidence that subtle modifications to ligands greatly influence their
pharmacological properties. Delineation of the biological activity
elicited by compounds with various chemical properties together with
characterization of the complex ER antagonist signaling is expected to
increase our understanding of how to target estrogen-dependent tumors in
a more optimal manner
Maximum Tolerated Dose and Pharmacokinetics of Paclitaxel Micellar in Patients with Recurrent Malignant Solid Tumours : A Dose-Escalation Study
Introduction: A water-soluble Cremophor EL-free formulation of paclitaxel, in which retinoic acid derivates solubilize paclitaxel by forming micelles (paclitaxel micellar), was studied for the first time in man to establish the maximum tolerated dose (MTD) and to characterize the pharmacokinetics (PK). Methods: This was an open-label, one-arm, dose-escalating study in patients with advanced solid malignant tumours, for which no standard therapy was available or had failed. Paclitaxel micellar was given as 1-h intravenous infusion every 21 days for 3 cycles, mainly without premedication. Plasma samples were collected during 24 h at the first cycle and paclitaxel concentrations were assayed by high-performance liquid chromatography. PK was evaluated using a two-compartment model. Results: Thirty-four patients received paclitaxel micellar at doses ranging between 90 and 275 mg/m2. MTD was established as 250 mg/m2. Fatigue and neuropathy were the most frequent dose-limiting toxicities. No hypersensitivity reactions were observed. PK of paclitaxel was evaluated in 25 data sets. Paclitaxel micellar had a rapid initial distribution phase, mean half-life 0.55 h, estimated to be completed 3 h after dosing and a mean terminal half-life of 8.8 h. Mean clearance was 13.4 L/h/m2 with fivefold interindividual variability. The residual areas after 10 h and 24 h were 15.7 ± 8.6% and 5.7 ± 3.9% of the area under the plasma concentration–time curve to infinite time (AUCinf), respectively. Conclusion: No new side effects unknown for paclitaxel were observed. Maximum plasma concentration (Cmax) and AUCinf showed a tendency to increase linearly with dose within the 150–275 mg/m2dose range. The possibility to administer paclitaxel micellar without steroid premedication makes it an attractive candidate for further studies in combination with immunotherapy. Trial Registration: EudraCT no: 2004-001821-54