Molecular characterization of the microsomal tamoxifen binding site

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.     

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia

Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3(-/-) mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3-/- animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.     

Characterization of a new mouse model for human apolipoprotein A-I/C-III/A-IV deficiency

Human data raised the possibility that coronary heart disease is associated with mutations in the apolipoprotein gene cluster APOA1/C3/A4 that result in multideficiency of cluster-encoded apolipoproteins and hypoalphalipoproteinemia. To test this hypothesis, we generated a mouse model for human apolipoprotein A-I (apoA-I)/C-III/A-IV deficiency. Homozygous mutants (Apoa1/c3/a4(-/-)) lacking the three cluster-encoded apolipoproteins were viable and fertile. In addition, feeding behavior and growth were apparently normal. Total cholesterol (TC), high density lipoprotein cholesterol (HDLc), and triglyceride levels in the plasma of fasted mutants fed a regular chow were 32% (P < 0.001), 17% (P < 0.001), and 70% (P < 0.01), respectively, those of wild-type mice. When fed a high-fat Western-type (HFW) diet, Apoa1/c3/a4(-/-) mice showed a further decrease in HDLc concentration and a moderate increase in TC, essentially in non-HDL fraction. The capacity of Apoa1/c3/a4(-/-) plasma to promote cholesterol efflux in vitro was decreased to 75% (P < 0.001), and LCAT activity was decreased by 38% (P < 0.01). Despite the very low total plasma cholesterol, the imbalance in lipoprotein distribution caused small but detectable aortic lesions in one-third of Apoa1/c3/a4(-/-) mice fed a HFW diet. In contrast, none of the wild-type mice had lesions. These results demonstrate that Apoa1/c3/a4(-/-) mice display clinical features similar to human apoA-I/C-III/A-IV deficiency (i.e., marked hypoalphalipoproteinemia) and provide further support for the apoa1/c3/a4 gene cluster as a minor susceptibility locus for atherosclerosis in mice.     

Poly(ADP-ribose) polymerase-1 inhibition prevents eosinophil recruitment by modulating Th2 cytokines in a murine model of allergic airway inflammation: a potential specific effect on IL-5

We recently used a murine model of allergic airway inflammation to show that poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in the pathogenesis of asthma-related lung inflammation. In this study, we show that PARP-1 inhibition, by a novel inhibitor (TIQ-A) or by gene deletion, prevented eosinophilic infiltration into the airways of OVA-challenged mice. Such impairment of eosinophil recruitment appeared to take place after IgE production. OVA challenge of wild-type mice resulted in a significant increase in IL-4, IL-5, IL-10, IL-13, and GM-CSF secretions. Although IL-4 production was moderately affected in OVA-challenged PARP-1(-/-) mice, the production of IL-5, IL-10, IL-13, and GM-CSF was completely inhibited in ex vivo OVA-challenged lung cells derived from these animals. A single TIQ-A injection before OVA challenge in wild-type mice mimicked the latter effects. The marked effect PARP-1 inhibition exerted on mucus production corroborated the effects observed on the Th2 response. Although PARP-1 inhibition by gene knockout increased the production of the Th1 cytokines IL-2 and IL-12, the inhibition by TIQ-A exerted no effect on these two cytokines. The failure of lung cells derived from OVA-challenged PARP-1(-/-) mice to synthesize GM-CSF, a key cytokine in eosinophil recruitment, was reestablished by replenishment of IL-5. Furthermore, intranasal administration of IL-5 restored the impairment of eosinophil recruitment and mucus production in OVA-challenged PARP-1(-/-) mice. The replenishment of either IL-4 or IgE, however, did not result in such phenotype reversals. Altogether, these results suggest that PARP-1 plays a critical role in eosinophil recruitment by specifically regulating the cascade leading to IL-5 production.     

Correlation between decreased sensitivity of the Daudi lymphoma cells to VP-16-induced apoptosis and deficiency in DNAS1L3 expression

A functional relationship between the apoptotic endonuclease DNAS1L3 and the chemotherapeutic drug VP-16 was established. The lymphoma cell line, Daudi, exhibited a significant resistance to VP-16 treatment in comparison to the lymphoma/leukemia cell line, U-937. While U-937 cells degraded their DNA into internucleosomal fragments, Daudi cells failed to undergo such fragmentation in response to the drug. Activation of both caspase-3 and DNA fragmentation factor was not sufficient to trigger internucleosomal DNA fragmentation in Daudi cells. No correlation was found between expression levels of topoisomerase-II, Pgp, Bcl-2, Bax, or Bad and decreased sensitivity of Daudi cells to VP-16. Daudi cells failed to express DNAS1L3 and ectopic expression of this protein significantly sensitized the cells to VP-16. An enhancement of caspase-3 activity and collapse of mitochondrial membrane potential underlie DNAS1L3-mediated sensitization of Daudi cells to VP-16, which may be a direct result of DNAS1L3-mediated increase in PARP-1-activating DNA breaks after VP-16 treatment. Our results suggest that DNAS1L3 plays an active role in lymphoma cell sensitization to VP-16 and that its deficiency may constitute a novel mechanism of drug resistance in these cells.     

Reversible inactivation of the transcriptional function of P53 protein by farnesylation

Background  

The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function.     

Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damages

Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αβ T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1β and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.