Mitochondrial DNA sequence divergence among big cats and their hybrids

Mitochondrial DNA sequence variation was used to distinguish between various big catspecies and their hybrids. Mitochondrial D loop sequencing revealed the presence of only one haplotype among the Asiatic lions while the hybrids (Asiatic×African) exhibited a number of haplotypes, which showed homology with the African lion sequence. this strongly indicates the population. the sequence divergence reveals the two subspecies must have split about 80,000 to 100,000 years ago. Similar analysis also helped in distinguishing between Indian and Siberian tigers. Microsatellite studies had earlier shown the presence of siberian tiger alleles in the Dhudhwa tiger reserve population. Mitochondrial cytochrome b gene sequence analysis of the hair samples from suspected Indian and Siberian tigerhybrids of the Dhudhwa tiger reserve revealed the presence of Indian tiger mitochondrial DNA haplotype

Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases.

BACKGROUND: Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics. METHODS: We used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development. RESULTS: We confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition. CONCLUSION: PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment

Transcriptional regulation of 12/15-lipoxygenase expression and the implication of the enzyme in hepoxilin biosynthesis and apoptosis

Die 12/15-Lipoxygenasen (12/15-LOXs) gehören zu einer heterogenen Klasse Lipid-peroxidierender Enzyme, deren biologische Rolle noch nicht vollständig geklärt ist. Eine Reihe experimenteller Daten deuten darauf hin, dass diese Enzym an Reifungs- und Differenzierungsprozessen beteiligt sind und auch für die Pathogenese verschiedener Erkrankungen (Asthma bronchiale, Entzündung, Atherosklerose) bedeutsam zu sein scheinen. Die Expression von 12/15-LOXs wird in vielen Säugetierzellen durch TH2-Zytokine reguliert und die Zytokin-induzierte Signaltransduktionskaskade verläuft über die Aktivierung van JAK-Kinasen und STAT6. Nach einer Stimulation von A549 Lungenkarzinomzellen mit Interleukin-4 (IL-4) kommt es erst nach 12 Stunden zu einer Hochregulation der 12/15-LOX mRNA Expression. Untersuchungen zum Induktionsmechanismus haben gezeigt, dass Genistein, ein Hemmstoff von Tyrosinkinasen, die Phosphorylierung von STAT6 und dessen Bindung an den Promoter des 12/15-LOX Gens verhinderte. Damit konnte die Induktion der katalytisch aktiven LOX geblockt werden. In Gegensatz dazu verhinderte Zykloheximid, ein spezifischer Hemmstoff der Proteinbiosynthese, die Expression der 12/15-LOX mRNA nicht, Dieses Ergebnis deutet darauf hin, dass die Neusynthese eines Transkriptionsfaktors im Rahmen der IL-4 induzierten Transduktionskaskade unwahrscheinlich ist. Weiterhin wurde beobachtet, dass IL-4 die zelluläre Histonacetyltransferase-Aktivität stark erhöhte und dass dieser Effekt überwiegend auf die enzymatische Aktivität des (CREB-bindenden Protein)-bindenden Proteins (CBP) zurückzuführen ist. Transfektion der Zellen mit E1A, einem viralen Protein, welches als Hemmstoff der Histonacetyltransferase-Aktivität von CBP/p300 bekannt ist, führte zu einer Unterdrückung der 12/15-LOX Expression. Andererseits stimuliert Natriumbutyrat, ein Hemmstoff der Histondeacetylase, die 12/15-LOX Synthese. Damit konnte gezeigt werden, dass die Acetylierung von Histonproteinen und von STAT6 ein essentieller Prozesse bei der IL-4 induzierten 12/15-LOX Expression in A549 Zellen ist. Weiterhin belegen diese Daten, dass sowohl die Phosphorylierung als auch die Acetylierung von STAT6 an der transkriptionellen Aktivierung des 12/15-LOX Gens beteiligt sind, obwohl beide Prozesse eine unterschiedliche Kinetik aufweisen. STAT6 Phosphorylierung erfolgt innerhalb der ersten Stunde nach IL-4 Stimulation, während die Acetylierungsreaktion zeitlich verzögert abläuft. Zusammenfassend kann die Signaltransduktionskaskade, die in A549 Zellen zur Expression der 12/15-LOX führt, wie folgt beschrieben werden: IL-4 induziert über die Aktivierung von JAK-Kinasen eine Phosphorylierung von STAT6, dessen Bindung an den 12/15-LOX Promotor jedoch zunächst durch nicht-acetylierte Histonproteine verhindert wird. Nach 9-11 Stunden werden Histone und der phosphorylierte STAT6 durch die Acetyltransferase-Aktivität von CBP/p300 acetyliert. Diese Reaktion führt zu einer Veränderung der Histonstruktur, wodurch die Bindung von modifizierten STAT6 und damit die Expression des 12/15-LOX Gens ermöglicht wird. Als wesentliche zellphysiologische Konsequenz der IL-4 induzierten 12/15-LOX Expression in A549 Zellen, wurde eine Apoptoseinduktion beobachtet. Das endogene 12/15-LOX Produkt 15-HETE bindet an den Kernrezeptor PPARg und induziert damit den programmierten Zelltod. Vorinkubation von A549 Zellen mit dem LOX-Hemmstoff NDGA oder der Einsatz von PPARg Dominant Negativ Vektor verhinderten die Apoptose. Mechanistische Untersuchungen zum Ablauf des durch IL-4 induzierten Zelltodes zeigten, dass der Prozess überwiegend dem extrinsischen Mechanismus folgt, bei dem Kaspasen-8 direkt zu einer Aktivierung der Effektorkaspase-3 führt. Der mitochondriale Mechanismus (Spaltung von Bid bzw. initiale Cytochrom C Freisetzung) scheint dabei nicht involviert zu sein. Die IL-4 induzierte Apoptose könnte von patho-physiologischer Bedeutung für den Verlauf von Lungenerkrankungen sein, bei denen Zellen mit hoher konstitutiver 12/15-LOX Expression, z.B. eosinophile Granulozyten, beteiligt sind. Hepoxiline sind bioaktive Mediatoren des 12/15-LOX Weges der Arachidonsäurekaskade, die durch Isomerisierung des primären Oxygenierungsproduktes 12S-HpETE gebildet werden. Zu Beginn unserer Untersuchungen war überwiegend unklar, welche Enzyme an der Isomerisierungsreaktion beteiligt sind. Bei der Suche nach geeigneten zellulären Modellen für die Untersuchung dieser Fragestellung fanden wir heraus, dass in den Ratteninsulinom-Zellen Rinm5F, die wegen ihres Mangels an Glutathionperoxidasen eine geringe Kapazität zur Reduktion von 12S-HpETE aufweisen, die Synthese von Hepoxilin A3 (HXA3) besonders hoch ist. Da wir vermuteten, dass 12/15-LOXs für die Isomerisierung von 12S-HpETE zu HXA3 verantwortlich sein könnten, verfolgten wir eine duale Forschungsstrategie um experimentelle Hinweise für unsere Arbeitshypothese zu finden. In den 12/15-LOX exprimierenden Rinm5F Zellen führte eine Immunopräzipitation mit 12/15-LOX spezifischen Antikörper zu einen vollständigen Verlust der 12/15-LOX- und der Hepoxilinsynthase-Aktivität eines Zelllysates. Beide Aktivitäten wurden fast vollständig im Immunopräzipitat wiedergefunden. 2. Transfektion von HeLa Zellen, die selbst keine 12/15-LOX exprimieren, mit 12/15-LOX und gleichzeitige Hemmung der zellulären Glutathionperoxidasen (Depletion von GSH mit Diethlmaleat) führte zu einer deutlichen zellulären Hepoxilinsynthese. Bei entsprechenden Kontrolltransfektanten wurde diese Aktivität nicht beobachtet. Weiterhin konnte festgestellt werden, dass rekombinante 12/15-LOXs (Expression in E. coli) in vitro eine intrinsische Hepoxinsynthase-Aktivität aufweisen, wenn 12S-HpETE als Substrat angeboten wird. Diese Daten belegen, dass 12/15-LOXs neben den bisher beschriebenen katalytischen Aktivitäten (Oxygenase, Hydroperoxidase, Leukotrienesynthase) auch Hepoxilinsynthase-Aktivität aufweisen.12/15-Lipoxygenases (human 15-LOX-1, rat 12/15-lipoxygenase) belong to family of lipid peroxidising enzymes. The enzyme has been implicated with roles in a variety of pathological conditions such as asthma, atherosclerosis, inflammation and in cellular differentiation. The enzyme expression in most human cell types is tightly regulated by Th2 cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13). Interleukin-4 (IL-4) induces expression of reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) in various mammalian cells via the Janus kinase/signal transducer and activator of transcription 6 (STAT6) signaling system. 15-LOX-1 mRNA expression was first observed only 12h post IL-4 stimuation and required a minimum of 11h exposure to the cytokine. The mechanism of 15-LOX-1 induction in A549 lung epithelial cells and the observed delay was studied and it was found that genistein, a potent tyrosine kinase inhibitor, prevented phopsphorylation of STAT6, its binding to the 15-LOX-1 promoter, and the expression of catalytically active enzyme. In contrast, cycloheximide did not prevent 15-LOX-1 induction. Surprisingly, it was observed that IL-4 up-regulated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300, which is responsible for acetylation of nuclear histones and STAT6. The acetylation of both proteins appears to be essential for the IL-4-induced signal transduction cascade, because inhibition of CBP/p300 by the viral wild-type E1A oncoprotein abrogated acetylation of both histones and STAT6 and strongly suppressed transcriptional activation of the 15-LOX-1 gene. Moreover, the inhibition by sodium butyrate of histone deacetylases, which apparently suppress 15-LOX-1 gene transcription, synergistically enhanced the IL-4-stimulated 15-LOX-1 expression. These data suggest that both phosphorylation and acetylation of STAT6 as well as acetylation of nuclear histones are involved in transcriptional activation of the 15-LOX-1 gene, although these reactions follow differential kinetics. STAT6 phosphorylation proceeds within the first hour of IL-4 stimulation. In contrast, CBP/p300-mediated acetylation requires 9-11 h, and similar kinetics were observed for the expression of the active enzyme. Thus, the results suggest that in the absence of IL-4, nuclear histones may be bound to regulatory elements of the 15-LOX-1 gene, preventing its transcription. IL-4 stimulation causes rapid phosphorylation of STAT6, but its binding to the promoter appears to be prevented by nonacetylated histones. After 9-11 h, when histones become acetylated, STAT6 binding sites may be demasked so that the phosphorylated and acetylated transcription factor can bind to activate gene transcription. The proinflammatory cytokine IL-4 is secreted in large amounts during allergic inflammatory response in asthma and plays a pivotal role in the airway inflammation. IL-4 has been shown to up-regulate 15-lipoxygenase and produce 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in A549 cells via the Janus kinase/STAT6 pathway under coactivation of CREB binding protein/p300. IL-4 has also been shown to up-regulate peroxisome proliferator-activated receptor (PPARg ) nuclear receptors in macrophages and A549 cells. In this study it is observed that 15(S)-HETE binds to PPARg nuclear receptors and induces apoptosis in A549 cells. Moreover, pre-treatment of cells with nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented PPARg activation and apoptosis. The latter was accomplished by the interaction of the 15(S)-HETE/PPARg complex with the adapter protein Fas-associating protein with death domain and caspase-8, as shown by transfection of Fas-associating protein with death domain dominant negative vector and cleavage of caspase 8 to active subunits p41/42 and p18. Whereas IL-4 and PPARg ligands failed to induce cleavage of Bid and release of cytochrome c from mitochondria, they caused translocation of the proapoptotic protein Bax from cytoplasm to mitochondria with a concomitant decrease in the Bcl-XL level. The cells were, thereofre, observed to follow the extrinsic pathway of apoptosis where caspase-8 directly activates the effector caspase-3, bypassing the mitochondria. The data also suggests that in IL-4-stimulated cells the 15(S)-HETE/PPARg complex down-regulates Bcl-XL, and the translocation of Bax to the mitochondria commits the cell to apoptosis. The IL-4-induced apoptosis may contribute to severe loss of alveolar structures and infiltration of eosinophils, mononuclear phagocytes, etc., into the lung tissue as observed in chronic asthma patients. The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of hepoxilins. Interestingly, rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), were observed to produce solely hepoxilin A3 (HXA3). Since HXA3 synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, suggesting that a HXA3 synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption AA was incubated with HeLa cells overexpressing the rat 12/15-LOX. Neither HXA3 nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA3 synthase and 12-LOX activities. Moreover, recombinant rat 12/15-LOX produced HXA3 when incubated with 12-HpETE. Further confirmation was obtained by immunoprecipitation with 12/15-LOX specific antibodies. Immunoprecipitation of Rinm5F lysates results in the depletion of hepoxilin synthase activity. The hepoxilin synthase activity was localised in the immunoprecipitated protein. Thus, cells containing rat 12/15-LOX also possess an intrinsic HXA3 synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA3 is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as Rinm5F. Furthermore, formation of corresponding epoxyhydroxy products was observed when 15-HpETE was used as substrate, indicating a broad range of specificity for the enzyme

Genetic variation in Asiatic lions and Indian tigers

Previous reports have suggested that Asiatic lions and tigers are highly inbred and exhibit very low levels of genetic variation. Our analyses on these species have shown much higher degrees of polymorphism than reported. Randomly amplified polymorphic DNA (RAPD) analysis of 38 Asiatic lions, which exist as a single population in the Gir Forest Sanctuary in India, shows an average heterozygosity of 25.82% with four primers. Sperm motility studies by our colleagues corroborate this data. In Indian tigers, microsatellite analysis of five CA repeat loci and multilocus fingerprinting using Bkm 2(8) probe on a population of 22 individuals revealed a heterozygosity of 22.65%. Microsatellite analysis at loci Fca 77 and Fca 126 revealed polymorphism amongst the Asiatic X African lion hybrids, which has enabled us to use these as markers to discriminate the pure Asiatic lions from the hybrids. A similar analysis was used to identify hybrids of Indian and Siberian tigers through polymerase chain reaction (PCR) amplification of hair samples. To ascertain the variation which existed before the population bottleneck at the turn of the present century, microsatellite analysis was performed on 50- to 125-year-old skin samples from museum specimens. Our results show similar levels of genetic variability as in the present population (21.01%). This suggests that low genetic variability may be the characteristic feature of these species and not the result of intensive inbreeding. DNA fingerprinting studies of Asiatic lions and tigers have helped in identifying individuals with high genetic variability which can be used for conservation breeding programs

HDAC inhibitors induce apoptosis in glucocorticoid-resistant acute lymphatic leukemia cells despite a switch from the extrinsic to the intrinsic death pathway.

Inhibitors of histone deacetylases (HDACi's) are promising novel tools for cancer therapy. We have compared the growth inhibitory and apoptogenic potential of the pan-HDACi SAHA and the sub-class I selective HDAC inhibitor MS275, as well as valproic acid (VPA) on glucocorticoid sensitive and resistant B (B-ALL) and T (T-ALL) cell acute lymphoblastic leukemia cells and patients blasts. In contrast, to our previous results with U937 acute myeloid leukemia (AML) cells which showed a similar activity of MS275 and SAHA in growth inhibition and apoptosis induction, both B and T-ALL cells were much more efficiently killed by SAHA and VPA than by MS275. The same relative potency was observed with some patient ALL blasts treated ex vivo. SAHA displayed similar efficacy on glucocorticoid-sensitive and insensitive ALL cells but did not synergize with dexamethasone. In studying mediators of apoptosis we found that the TRAIL receptor DR5 is constitutively expressed in glucocorticoid-sensitive CEM-C7 cells which are also TRAIL sensitive. In contrast, glucocorticoid-insensitive CEM-C1 cells do not express DR5 and are insensitive to TRAIL. However, SAHA induces, in addition to p21(WAF1/CIP1) also re-expression of DR5. Importantly, SAHA-induced apoptosis of CEM-C7 cells operates through initiator caspase 10, while it induces apoptosis of CEM-C1 cells through the intrinsic, as well as through caspase-independent death pathways. Our data suggest that the generation of resistance to glucocorticoids has dramatically altered death signaling in these cells and that SAHA overcomes these restrictions by inducing alternative death pathways

Recruitment of RXR by Homotetrameric RARα Fusion Proteins Is Essential for Transformation

SummaryWhile formation of higher-order oncogenic transcriptional complexes is critical for RARα fusion proteins in acute promyelocytic leukemia, the essential components and their roles in mediating transformation are still largely unknown. To this end, the present study demonstrates that homodimerization is not sufficient for RARα fusion-mediated transformation, which requires higher-order homotetramerization. Surprisingly, intrinsic homo-oligomeric DNA binding by the fusion proteins is also dispensable. Importantly, higher-order RXR/RARα fusion hetero-oligomeric complexes that aberrantly recruit transcriptional corepressors to downstream targets are essential for transformation. Intervention of RXR-dependent pathways by panRXR-agonists or RXRα shRNAs suppresses RARα fusion-mediated transformation. Taken together, these results define the oncogenic threshold for self-association and reveal the pathological significance of higher-order RARα fusion/RXR hetero-oligomeric complexes and their potential value as a therapeutic target

Growth factor-antagonized rexinoid apoptosis involves permissive PPARgamma/RXR heterodimers to activate the intrinsic death pathway by NO.

International audienceGrowth factor (GF) deprivation and/or blocking of cognate signaling can induce apoptosis and is the basis of several cancer treatment paradigms. We observed that RXR agonists (rexinoids) induce apoptosis of tumor cells when GF support is abrogated. This "rexinoid apoptosis" involves activation of both iNOS and eNOS by RXR-PPARgamma and results in production of apoptogenic NO. IGF/EGF-induced IGF receptor 1-mediated MAP kinase blocks rexinoid apoptosis by RXR phosphorylation. Combining rexinoids with the MAPK inhibitor U0126 induced apoptosis in human cancer cells in vitro and ex vivo and blocked xenograft growth in vivo. Our results suggest a regulatory mechanism in which GF signaling antagonizes RXR-PPARgamma-mediated default apoptosis to sustain cell life