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Marilena Manea
; Ulrike Leurs
; Erika Orban
; Zsuzsa Baranyai
; Peter Öhlschläger
; Andreas Marquardt
; Akos Schulcz
; Miguel Tejeda
Raphaëlle Luisier
; Harri Lempiäinen
; Nina Scherbichler
; Albert Braeuning
; Miriam Geissler
; Valerie Dubost
; Arne Müller
; Nico Scheer
; Salah-Dine Chibout
; Hisanori Hara
; Frank Picard
; Diethilde Theil
; Philippe Couttet
; Antonio Vitobello
; Olivier Grenet
; Bettina Grasl-Kraupp
; Heidrung Ellinger-Ziegelbauer
; John P. Thomson
; Richard R. Meehan
; Clifford R. Elcombe
; Colin J. Henderson
; C. Roland Wolf
; Michael Schwarz
; Pierre Moulin
; Remi Terranova
; Jonathan G. Moggs
The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CARᴷᴼ-PXRᴷᴼ), double humanized CAR and PXR (CARʰ-PXRʰ), and wild-type C57BL/6 mice. Wild-type and CARʰ-PXRʰ mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CARᴷᴼ-PXRᴷᴼ mouse livers and largely reversible in wild-type and CARʰ-PXRʰ mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CARʰ-PXRʰ mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.
Simon Lindner
; René Burger
; Douglas N. Rutledge
; Xuan Tung Do
; Jessica Rumpf
; Bernd W. K. Diehl
; Margit Schulze
; Yulia Monakhova
Although several successful applications of benchtop nuclear magnetic resonance (NMR) spectroscopy in quantitative mixture analysis exist, the possibility of calibration transfer remains mostly unexplored, especially between high- and low-field NMR. This study investigates for the first time the calibration transfer of partial least squares regressions [weight average molecular weight (Mw) of lignin] between high-field (600 MHz) NMR and benchtop NMR devices (43 and 60 MHz). For the transfer, piecewise direct standardization, calibration transfer based on canonical correlation analysis, and transfer via the extreme learning machine auto-encoder method are employed. Despite the immense resolution difference between high-field and low-field NMR instruments, the results demonstrate that the calibration transfer from high- to low-field is feasible in the case of a physical property, namely, the molecular weight, achieving validation errors close to the original calibration (down to only 1.2 times higher root mean square errors). These results introduce new perspectives for applications of benchtop NMR, in which existing calibrations from expensive high-field instruments can be transferred to cheaper benchtop instruments to economize.
Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [4Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH2) containing two lysine residues in positions 4 and 8, whose ϵ-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2) was used as a scaffold; an additional lysine residue was coupled to the ϵ-amino group of 8Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.
Harri Lempiäinen
; Philippe Couttet
; Federico Bolognani
; Arne Müller
; Valérie Dubost
; Raphaëlle Luisier
; Alberto del Rio-Espinola
; Veronique Vitry
; Elif B. Unterberger
; John P. Thomson
; Fridolin Treindl
; Ute Metzger
; Clemens Wrzodek
; Florian Hahne
; Tulipan Zollinger
; Sarah Brasa
; Magdalena Kalteis
; Magali Marcellin
; Fanny Giudicelli
; Albert Braeuning
; Laurent Morawiec
; Natasa Zamurovic
; Ulrich Längle
; Nico Scheer
; Dirk Schübeler
; Jay Goodman
; Salah-Dine Chibout
; Jennifer Marlowe
; Dietlinde Theil
; David J. Heard
; Olivier Grenet
; Andreas Zell
; Markus F. Templin
; Richard R. Meehan
; Roland C. Wolf
; Clifford R. Elcombe
; Michael Schwarz
; Pierre Moulin
; Rémi Terranova
; Jonathan G. Moggs
The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, sug- gesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.
Nathan D. Lawson
; Nico Scheer
; Van N. Pham
; Ceol-Hee Kim
; Ajay B. Chitnis
; José A. Campos-Ortega
; Brant M. Weinstein