The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway

Dexfenfluramine and the oestrogen-metabolizing enzyme CYP1B1 in the development of pulmonary arterial hypertension

<p>Aims: Pulmonary arterial hypertension (PAH) occurs more frequently in women than men. Oestrogen and the oestrogen-metabolising enzyme cytochrome P450 1B1 (CYP1B1) play a role in the development of PAH. Anorectic drugs such as dexfenfluramine (Dfen) have been associated with the development of PAH. Dfen mediates PAH via a serotonergic mechanism and we have shown serotonin to up-regulate expression of CYP1B1 in human pulmonary artery smooth muscle cells (PASMCs). Thus here we assess the role of CYP1B1 in the development of Dfen-induced PAH.</p> <p>Methods and results: Dfen (5 mg kg−1 day−1 PO for 28 days) increased right ventricular pressure and pulmonary vascular remodelling in female mice only. Mice dosed with Dfen showed increased whole lung expression of CYP1B1 and Dfen-induced PAH was ablated in CYP1B1−/− mice. In line with this, Dfen up-regulated expression of CYP1B1 in PASMCs from PAH patients (PAH-PASMCs) and Dfen-mediated proliferation of PAH-PASMCs was ablated by pharmacological inhibition of CYP1B1. Dfen increased expression of tryptophan hydroxylase 1 (Tph1; the rate-limiting enzyme in the synthesis of serotonin) in PAH-PASMCs and both Dfen-induced proliferation and Dfen-induced up-regulation of CYP1B1 were ablated by inhibition of Tph1. 17β-Oestradiol increased expression of both Tph1 and CYP1B1 in PAH-PASMCs, and Dfen and 17β-oestradiol had synergistic effects on proliferation of PAH-PASMCs. Finally, ovariectomy protected against Dfen-induced PAH in female mice.</p> <p>Conclusion: CYP1B1 is critical in the development of Dfen-induced PAH in mice in vivo and proliferation of PAH-PASMCs in vitro. CYP1B1 may provide a novel therapeutic target for PAH.</p&gt

Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.

Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug

Anticonvulsant and analgesic activities of crude extract and its fractions of the defensive secretion from the Mediterranean sponge, Spongia officinalis.

International audienceABSTRACT: This study progresses in the direction of identifying component(s) from the Mediterranean sponge, Spongia officinalis with anticonvulsant and analgesic activities. We investigated the efficacy of crude extract and its semi-purified fractions (F1-F3) of the defensive secretion from Spongia officinalis for their in vivo anticonvulsant activity using the pentylenetetrazole (PTZ) seizure model and analgesic activity using the writhing test in mice. Among the series the crude extract exhibited interesting analgesic activity in a dose dependent manner. Similarly the fraction F2 showed a partial protection of mice from PTZ-induced seizure and interesting analgesic activity in a dose dependent manner. The purification and the determination of chemical structure(s) of compound(s) of this active fraction are under investigation

Combined targeting of MDM2 and CDK4 is synergistic in dedifferentiated liposarcomas

Abstract Purpose MDM2 and CDK4 are frequently co-amplified in well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). We aimed to determine whether combined MDM2/CDK4 targeting is associated with higher antitumour activity than a single agent in preclinical models of DDLPS. Experimental design DDLPS cells were exposed to RG7388 (MDM2 antagonist) and palbociclib (CDK4 inhibitor), and apoptosis and signalling/survival pathway perturbations were monitored by flow cytometry and Western blotting. Xenograft mouse models were used to assess tumour growth and survival. Treatment efficacy was assessed by Western blotting, histopathology and tumour volume. Results RG7388 and palbociclib together exerted a greater antitumour effect than either drug alone, with significant differences in cell viability after a 72-h treatment with RG7388 and/or palbociclib. The combination treatment significantly increased apoptosis compared to the single agents. We then analysed the in vivo antitumour activity of RG7388 and palbociclib in a xenograft model of DDLPS. The combination regimen reduced the tumour growth rate compared with a single agent alone and significantly increased the median progression-free survival. Conclusions Our results provide a strong rationale for evaluating the therapeutic potential of CDK4 inhibitors as potentiators of MDM2 antagonists in DDLPS and justify clinical trials in this setting