Circulating CASK is associated with recurrent focal segmental glomerulosclerosis after transplantation.

INTRODUCTION: Focal and Segmental GlomeruloSclerosis (FSGS) can cause nephrotic syndrome with a risk of progression to end-stage renal disease. The idiopathic form has a high rate of recurrence after transplantation, suggesting the presence of a systemic circulating factor that causes glomerular permeability and can be removed by plasmapheresis or protein-A immunoadsorption. RESULTS: To identify this circulating factor, the eluate proteins bound on therapeutic immunoadsorption with protein-A columns were analyzed by comparative electrophoresis and mass spectrometry. A soluble form of calcium/calmodulin-dependent serine protein kinase (CASK) was identified. CASK was immunoprecipitated only in the sera of patients with recurrent FSGS after transplantation and not in control patients. Recombinant-CASK (rCASK) induced the reorganization of the actin cytoskeleton in immortalized podocytes, a redistribution of synaptopodin, ZO-1,vinculin and ENA. rCASK also induced alterations in the permeability of a monolayer of podocytes and increased the motility of pdodocytes in vitro. The extracellular domain of CD98, a transmembrane receptor expressed on renal epithelial cells, has been found to co-immunoprecipitated with rCASK. The invalidation of CD98 with siRNA avoided the structural changes of rCask treated cells suggesting its involvement in physiopathology of the disease. In mice, recombinant CASK induced proteinuria and foot process effacement in podocytes. CONCLUSION: Our results suggest that CASK can induce the recurrence of FSGS after renal transplantation.

Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis.

In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

Unsaturated fatty acids induce non-canonical autophagy.

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1(+/-) mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.

The captured retroviral envelope syncytin-A and syncytin-B genes are conserved in the Spalacidae together with hemotrichorial placentation.

Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Multiple independent events of syncytin gene capture were found to have occurred in primates, rodents, lagomorphs, carnivores, and ruminants. In the mouse, two syncytin-A and -B genes are present, which trigger the formation of the two-layered placental syncytiotrophoblast at the maternal-fetal interface, a structure classified as hemotrichorial. Here, we identified syncytin-A and -B orthologous genes in the genome of all Muroidea species analyzed, thus dating their capture back to about at least 40 million years ago, with evidence that they evolved under strong purifying selection. We further show, in the divergent Spalacidae lineage (blind mole rats [Spalax]), that both syncytins have conserved placenta-specific expression, as revealed by RT-PCR analysis of a panel of Spalax galili tissues, and display fusogenic activity, using ex vivo cell-cell fusion assays. Refined analysis of the placental architecture and ultrastructure revealed that the Spalax placenta displays a hemotrichorial organization of the interhemal membranes, as similarly observed for other Muroidea species, yet with only one trophoblastic cell layer being clearly syncytialized. In situ hybridization experiments further localized syncytin transcripts at the level of these differentiated interhemal membranes. These findings argue for a role of syncytin gene capture in the establishment of the original hemotrichorial placenta of Muroidea, and more generally in the diversity of placental structures among mammals.

The HERV-K human endogenous retrovirus envelope protein antagonizes Tetherin antiviral activity.

UNLABELLED: Endogenous retroviruses are the remnants of past retroviral infections that are scattered within mammalian genomes. In humans, most of these elements are old degenerate sequences that have lost their coding properties. The HERV-K(HML2) family is an exception: it recently amplified in the human genome and corresponds to the most active proviruses, with some intact open reading frames and the potential to encode viral particles. Here, using a reconstructed consensus element, we show that HERV-K(HML2) proviruses are able to inhibit Tetherin, a cellular restriction factor that is active against most enveloped viruses and acts by keeping the viral particles attached to the cell surface. More precisely, we identify the Envelope protein (Env) as the viral effector active against Tetherin. Through immunoprecipitation experiments, we show that the recognition of Tetherin is mediated by the surface subunit of Env. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal; therefore, it uses a yet-undescribed mechanism to inactivate Tetherin. We also assessed all natural complete alleles of endogenous HERV-K(HML2) Env described to date for their ability to inhibit Tetherin and found that two of them (out of six) can block Tetherin restriction. However, due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population, and it is likely that individuals will not all possess the same anti-Tetherin potential. Because of Tetherin's role as a restriction factor capable of inducing innate immune responses, this could have functional consequences for individual responses to infection. IMPORTANCE: Tetherin, a cellular protein initially characterized for its role against HIV-1, has been proven to counteract numerous enveloped viruses. It blocks the release of viral particles from producer cells, keeping them tethered to the cell surface. Several viruses have developed strategies to inhibit Tetherin activity, allowing them to efficiently infect and replicate in their host. Here, we show that human HERV-K(HML2) elements, the remnants of an ancient retroviral infection, possess an anti-Tetherin activity which is mediated by the envelope protein. It is likely that this activity was an important factor that contributed to the recent, human-specific amplification of this family of elements. Also, due to their recent amplification, HERV-K(HML2) elements are highly polymorphic in the human population. Since Tetherin is a mediator of innate immunity, interindividual variations among HERV-K(HML2) Env genes may result in differences in immune responses to infection.

DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+) T cells infected with HIV.

Productive HIV infection of CD4(+) T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

Preferential killing of p53-deficient cancer cells by reversine.

Reversine is a small synthetic molecule that inhibits multiple mitotic kinases, including MPS1 as well as Aurora kinase A and B (AURKA and AURKB). Here, we investigated the effects of reversine on p53-deficient vs p53-proficient cancer cells. We found that low doses (~0.5 µM) of reversine, which selectively inhibit MPS1 and hence impair the spindle assembly checkpoint, kill human TP53 (-/-) colon carcinoma cells less efficiently than their wild-type counterparts. In sharp contrast, high doses (~5 µM) of reversine induced hyperploidization and apoptosis to a much larger extent in TP53 (-/-) than in TP53 (+/+) cells. Such a selective cytotoxicity could not be reproduced by the knockdown of MPS1, AURKA and AURKB, neither alone nor in combination, suggesting that it involves multiple (rather than a few) molecular targets of reversine. Videomicroscopy-based cell fate profiling revealed that, in response to high-dose reversine, TP53 (-/-) (but not TP53 (+/+) ) cells undergo several consecutive rounds of abortive mitosis, resulting in the generation of hyperpolyploid cells that are prone to succumb to apoptosis upon the activation of mitotic catastrophe. In line with this notion, the depletion of anti-apoptotic proteins of the BCL-2 family sensitized TP53 (-/-) cells to the toxic effects of high-dose reversine. Moreover, the knockdown of BAX or APAF-1, as well as the chemical inhibition of caspases, limited the death of TP53 (-/-) cells in response to high-dose reversine. Altogether, these results suggest that p53-deficient cells are particularly sensitive to the simultaneous inhibition of multiple kinases, including MPS1, as it occurs in response to high-dose reversine.

Selective killing of p53-deficient cancer cells by SP600125.

The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53(+/+) and TP53(-/-) human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53(-/-) (but not TP53(+/+) ) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53(-/-) cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53(-/-) cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.

Autophagic removal of micronuclei.

Macroautophagy is known to participate in the quality control and turnover of cytoplasmic organelles, yet there is little evidence that macroautophagy targets nuclei in mammalian cells. Here, we investigated whether autophagy may target micronuclei, which arise as a result of deficient bipolar chromosome segregation in cells exposed to cell cycle perturbations. After removal of several distinct cell cycle blockers (nocodazole, cytochalasin D, hydroxyurea or SP600125), cells manifested an increase in the frequency of micronuclei (positive for histone H2B-RFP) as well as an increase in autophagic puncta (positive for GFP-LC3) over several days. A small but significant percentage of micronuclei co-localized with GFP-LC3 in autophagy-competent cells and this co-localization was lost after knockdown of ATG5 or ATG7. Electron microscopy analyses confirmed autophagic sequestration of micronuclei. "Autophagic micronuclei" (GFP-LC3⁺) were also decorated with p62/SQSTM1, while non-autophagic (GFP-LC3⁻) micronuclei where p62/SQSTM1 negative. In addition, GFP-LC3⁺ micronuclei exhibited signs of envelope degradation and γH2AX⁺ DNA damage foci, yet stained less intensively for chromatin markers, whereas GFP-LC3⁻ micronuclei were surrounded by an intact envelope and rarely exhibited markers or DNA damage. These results indicate that micronuclei can be subjected to autophagic degradation. Moreover, it can be speculated that removal of micronuclei may contribute to the genome-stabilizing effects of autophagy.

Inhibition of autophagy by TAB2 and TAB3.

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFß-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

A pair of co-opted retroviral envelope syncytin genes is required for formation of the two-layered murine placental syncytiotrophoblast.

In most mammalian species, a critical step of placenta development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast layer fulfilling essential fetomaternal exchange functions. Key insights into this process came from the discovery of envelope genes of retroviral origin, the syncytins, independently acquired by the human (syncytin-1 and -2), mouse (syncytin-A and -B), and rabbit (syncytin-Ory1) genomes, with fusogenic properties and placenta-specific expression. We previously showed that mouse syncytin-A is essential for the formation of one of the two syncytiotrophoblast layers and for embryo survival. Here, we have generated syncytin-B KO mice and demonstrate that syncytin-B null placenta displays impaired formation of syncytiotrophoblast layer II (ST-II), with evidence of unfused apposed cells, and enlargement of maternal lacunae disrupting the placenta architecture. Unexpectedly, syncytin-B null embryos are viable, with only limited late-onset growth retardation and reduced neonate number. Microarray analyses identified up-regulation of the connexin 30 gene in mutant placentae, with the protein localized at the fetomaternal interface, suggesting gap junction-mediated compensatory mechanisms. Finally, double-KO mice demonstrate premature death of syncytin-A null embryos if syncytin-B is deleted, indicating cooperation between ST-I and ST-II. These findings establish that both endogenous retrovirus-derived syncytin genes contribute independently to the formation of the two syncytiotrophoblast layers during placenta formation, demonstrating a major role of retroviral gene capture, through convergent evolution, to generate multiple placental structures. Although some are absolutely required for completion of pregnancy, others are still amenable to "epigenetic" compensations, thus illustrating the complexity of the molecular machinery that developed during placental evolution.

Erlotinib antagonizes constitutive activation of SRC family kinases and mTOR in acute myeloid leukemia.

Tyrosine kinases such as SRC family kinases (SFKs) as well as the mammalian target of rapamycin (mTOR) serine/threonine kinase are often constitutively activated in acute myeloid leukemia (AML) and hence constitute potential therapeutic targets. Here we demonstrate that the epidermal growth factor receptor (EGFR) inhibitor erlotinib, which has previously been shown to mediate antiproliferative/cytotoxic off-target effects in myelodysplastic syndrome (MDS) and AML blasts, reduces SFK overactivation. Erlotinib induced an arrest in the G 1 phase of the cell cycle that, in cells with constitutive SFK activation, could be recapitulated by chemical inhibition of SFKs with 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-α]pyrimidin-4-amine (PP2). Moreover, erlotinib inhibited the phosphorylation of mTOR targets like p70 (SK6) , stimulated the maturation of the autophagic marker LC3 and promoted the formation of autophagosomes. Notably, PP2 and the mTOR inhibitor rapamycin had a similar cell cycle-arresting activity to erlotinib, but neither of these compounds alone induced significant levels of cell death. Altogether, these results suggest that the therapeutic off-target effect of erlotinib may be linked to, yet cannot be entirely explained by, the inhibition of oncogenic signaling via SFKs and mTOR. Thus, combination therapies with erlotinib and rapamycin might be beneficial for MDS and AML patients.

Neuroendocrine regulation of autophagy by leptin.

The satiety hormone leptin plays a cardinal role in the pathophysiology of obesity and diabetes. Here, we show that pharmacological autophagy inducers like rapamycin, spermidine and resveratrol can reduce leptin concentrations in the serum of mice and that genetic inactivation of the leptin/leptin receptor system leads to an increase in autophagy in peripheral tissues including skeletal muscle, heart and liver. Paradoxically, intravenous or intraperitoneal administration of recombinant leptin protein also induced autophagy in these tissues. Moreover, leptin stimulated canonical autophagy in cultured human or mouse cell lines, a phenomenon that was coupled to the activation of adenosine monophosphate-dependent kianse (AMPK), as well as the inhibition of mammalian target of rapamycin (mTOR), and that was confirmed by autophagic flux measurements. These results suggest that leptin plays an important role in the neuroendocrine control of autophagy, underscoring the existence of novel links between metabolic control and autophagic flux that warrant further in-depth investigation.

Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within paraspeckle nuclear bodies.

Paraspeckles (PSPs) are nuclear bodies associated with the retention in the nucleus of specific mRNAs. Two isoforms of a long noncoding RNA (NEAT1_v1/Menε and NEAT1_v2/Menß) are required for the integrity of PSPs. Here, we analyzed the molecular organization of PSPs by immuno- and in situ hybridization electron microscopy. Detection of the paraspeckle markers PSPC1 and P54NRB/NONO confirm the identity between PSPs and the previously described interchromatin granule-associated zones (IGAZs). High-resolution in situ hybridization of NEAT1 transcripts revealed a highly ordered organization of IGAZ/PSPs. Although the 3.7-kb NEAT1_v1 and the identical 5' end of the 22.7-kb NEAT1_v2 transcripts are confined to the periphery, central sequences of NEAT1_v2 are found within the electron-dense core of the bodies. Moreover, the 3' end of NEAT1_v2 also localize to the periphery, indicating possible architectures for IGAZ/PSPs. These results further suggest that the organization of NEAT1 transcripts constrains the geometry of these bodies. Accordingly, we observed in HeLa and NIH 3T3 cells that IGAZ/PSPs are elongated structures with a well-defined diameter. Our results provide new insight on the ability of noncoding RNAs to form subcellular structures.

The matrix protein of vesicular stomatitis virus binds dynamin for efficient viral assembly.

Matrix proteins (M) direct the process of assembly and budding of viruses belonging to the Mononegavirales order. Using the two-hybrid system, the amino-terminal part of vesicular stomatitis virus (VSV) M was shown to interact with dynamin pleckstrin homology domain. This interaction was confirmed by coimmunoprecipitation of both proteins in cells transfected by a plasmid encoding a c-myc-tagged dynamin and infected by VSV. A role for dynamin in the viral cycle (in addition to its role in virion endocytosis) was suggested by the fact that a late stage of the viral cycle was sensitive to dynasore. By alanine scanning, we identified a single mutation of M protein that abolished this interaction and reduced virus yield. The adaptation of mutant virus (M.L4A) occurred rapidly, allowing the isolation of revertants, among which the M protein, despite having an amino acid sequence distinct from that of the wild type, recovered a significant level of interaction with dynamin. This proved that the mutant phenotype was due to the loss of interaction between M and dynamin. The infectious cycle of the mutant virus M.L4A was blocked at a late stage, resulting in a quasi-absence of bullet-shaped viruses in the process of budding at the cell membrane. This was associated with an accumulation of nucleocapsids at the periphery of the cell and a different pattern of VSV glycoprotein localization. Finally, we showed that M-dynamin interaction affects clathrin-dependent endocytosis. Our study suggests that hijacking the endocytic pathway might be an important feature for enveloped virus assembly and budding at the plasma membrane.

IKK connects autophagy to major stress pathways.

Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

The IKK complex contributes to the induction of autophagy.

In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IkappaB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-kappaB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-kappaB or ablation of the p65/RelA NF-kappaB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-kappaB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-beta suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis.

The effectiveness of ischemic preconditioning (IP) against hepatic ischemia/reperfusion injury during human liver surgery is linked to decreased apoptotic cell death as well as preservation of the ATP content in liver tissue. Overproduction of Bcl-2 is reported in preconditioned organs. In human liver biopsies exhibiting steatosis and/or vascular injuries (mainly peliosis) induced by chemotherapy, we find that the expression of Bcl-2 in centrolobular and peliotic areas colocalizes with the autophagy protein Beclin 1 in IP livers. Increased expression of phosphorylated Bcl-2 in preconditioned livers is associated with decreased immunoprecipitation of Beclin 1 and increased expression of LC3-II. The increased number of autophagic vacuoles seen by electron microscopy confirmed that IP could trigger autophagy in chemotherapy-injured livers, probably to reduce the pro-inflammatory necrotic cell death of hepatocytes or endothelial cells and to increase ATP levels. Indeed, necrosis is less frequent (p = 0.04) in IP livers than in the others although no change in apoptosis as assessed by TUNEL assay or caspase-3, -8 and -9 expressions is observed. In conclusion, Bcl-2 and Beclin 1 could be major targets in the regulation of cell death during ischemia/reperfusion injury modulating autophagy to switch on/off necrosis and/or apoptosis.

Liver circadian clock, a pharmacologic target of cyclin-dependent kinase inhibitor seliciclib.

Circadian disruption accelerates malignant growth and shortens survival, both in experimental tumor models and cancer patients. In previous experiments, tumor circadian disruption was rescued with seliciclib, an inhibitor of cyclin-dependent kinases (CDKs). This effect occurred at a selective dosing time and was associated with improved antitumor activity. In the current study, seliciclib altered robust circadian mRNA expression of the clock genes Rev-erb alpha, Per2, and Bmal1 in mouse liver following dosing at zeitgeber time (ZT) 3 (i.e., 3 h after the onset of the 12 h light span), when mice start to rest, but not at ZT19, near the middle of the 12 h dark span, when mice are most active. However, liver exposure to seliciclib, as estimated by the liver area under the concentration x time curve (AUC), was approximately 80% higher at ZT19 than at ZT3 (p = 0.049). Circadian clock disruption was associated with increased serum liver enzymes and modified glycogen distribution in hepatocytes, as revealed by biochemical determinations and optic and electronic microscopy. The extent of increase in liver enzymes was most pronounced following dosing at ZT3, as compared to ZT19 (p < 0.04). Seliciclib further up-regulated the transcriptional activity of c-Myc, a cell cycle gene that promotes cell cycle entry and G1-S transition (p < 0.001), and down-regulated that of Wee1, which gates cell cycle transition from G2 to M (p < 0.001). These effects did not depend upon drug dosing time. Overall, the results suggest the circadian time of seliciclib delivery is more critical than the amount of drug exposure in determining its effects on the circadian clock. Seliciclib-induced disruption of the liver molecular clock could account for liver toxicity through the resulting disruption of clock-controlled detoxification pathways. Modifications of cell cycle gene expression in the liver likely involve other mechanisms. Circadian clocks represent relevant targets to consider for optimization of therapeutic schedules of CDK inhibitors.

Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy.

BACKGROUND/AIMS: Liver pathology induced by chemotherapy (steatosis or vascular injury) is known to increase the liver's sensitivity to ischemia/ reperfusion (I/R) injury, thereby increasing morbidity and mortality after liver resection. Our aim was to assess whether ischemic preconditioning (IP) reduces I/R injury to livers with chemotherapy-induced pathology. METHODS: We analyzed a series of livers from patients treated with chemotherapy for colorectal cancer who underwent IP (n=30) or not (n=31) before hepatectomy. All but one of the livers exhibited chemotherapy-induced steatosis and/ or peliosis before the I/R insult. RESULTS: Necrosis was less frequent (p=0.038) in livers with IP than in the others. IP had no influence on apoptosis as assessed by terminal transferase uridyl nick-end labeling (TUNEL) assay or caspase-3, -8 and -9 expression. IP induced a twofold increase in B-cell leukemia/ lymphoma 2 (Bcl-2; p<0.05), which was localized to hepatocytes of centrolobular and peliotic areas and colocalized with the autophagy protein beclin-1 in livers with IP, suggesting their coordinated role in autophagy. Increased expression of the phosphorylated Bcl-2 was observed in preconditioned livers and was associated with a decreased immunoprecipitation of beclin-1 and the increased expression of light chain 3 type II (LC3-II). The increased number of autophagic vacuoles seen by electron microscopy confirmed an association of autophagy in chemotherapy-injured livers following IP. However, the differences in protein expression were not reflected in postresection liver-injury tests or measure of patient morbidity. CONCLUSIONS: IP is associated with a reduction in necrosis of hepatocytes already damaged by chemotherapy and an activation of autophagy. Bcl-2 and beclin-1 could be major targets in the regulation of cell death during I/R injury.