Proteomic identification of C/EBPa multiprotein complex reveals that JNK1, an activator of C/EBPa is downregulated in patients with acute myeloid leukemia (AML)

Functional inactivation of the transcription factor CAAT Enhancer Binding Protein Alpha (C/EBPα) either by mutation or direct protein-protein interaction leads to acute myeloid leukemia (AML), whereas the activation of C/EBPα restores normal myeloid cell differentiation. We and others have shown that protein-protein interactions of C/EBPα play a pivotal role in myeloid differentiation and AML. In the present study we applied proteomics based mass spectrometry to identify C/EBPα interacting proteins on a proteome-wide scale. For this, the GST and GST-DNA binding domain of C/EBP (GST-DBD) was incubated with nuclear extracts of U937 cells. Interacting proteins separated by two-dimensional gel electrophoresis were identified by MALDI-TOF mass spectrometry. Using this approach we could identify PAK6, MADP-1, ZNF45 and the c-Jun N-terminal kinase 1 (JNK1) as C/EBPα interacting proteins. Since JNK1 activates c-Jun, the contra-player for C/EBPα, we hypothesized that the JNK1 and C/EBPα interaction might have some biological relevance. We could show that JNK1 binds to the DNA binding domain of C/EBP in GST-pull-down and to the C/EBPα in co-immunoprecipitation assays in-vitro and in-vivo respectively. JNK1 phosphorylates and increases the half life of the C/EBPα protein via inhibiting its ubiquitination and thereby enhances its transactivation and DNA binding activity. Furthermore, JNK mRNA expression as well as its kinase activity is lower in the AML patients in comparison to normal bone marrow mononuclear cells which implicates a possible mechanism of C/EBPα inactivation in certain acute myeloid leukemia subtypes. Thus our data suggest that a JNK activity is required for C/EBPα activation in myeloid cells and a loss of JNK regulated C/EBPα expression may contribute to leukemogenesis.Die funktionelle Inaktivierung des Transkriptionsfaktors CAAT Enhancer Binding Protein alpha (C/EBPα) entweder durch Mutation oder durch direkte Protein-Protein-Interaktion führt zu akuter myeloischer Leukämie (AML), wohingegen die Aktivierung von C/EBPα eine normale myeloische Differenzierung gewährleistet. Wir und andere konnten zeigen, dass Protein- Protein-Interaktionen von C/EBPα eine entscheidende Rolle in der myeloischen Differenzierung und AML spielen. In der vorliegenden Studie haben wir die auf Proteomik basierende Massenspektrometrie genutzt, um die mit C/EBPα interagierenden Proteine in einem Proteom-weiten Maßstab zu identifizieren. Zu diesem Zweck wurde ein Fusionsprodukt aus GST und der DNA-bindenden Domäne von C/EBPα mit Kernextrakten von U937 inkubiert. Mit C/EBPα interagierende Proteine wurden mittels zweidimensionaler Gelelektrophorese separiert und durch MALDI-TOF-Massenspektrometrie identifiziert. Mit diesem Ansatz konnten wir PAK6, MADP-1, ZNF45 und die c-jun N-terminale Kinase 1 (JNK1) als interagierende Proteine von C/EBPα identifizieren. JNK1 aktiviert c-jun, einen Gegenspieler von C/EBPα. Daher stellten wir die Hypothese auf, dass die Interaktion von JNK1 und C/EBPα eine biologische Relevanz haben könnte. Wir konnten mittels GST-Pull-Down-Experimenten in-vitro zeigen, dass JNK1 mit der DNA-bindenden Domäne von C/EBP interagiert. Die Interaktion von JNK1 und C/EBPα konnte in-vivo durch Koimmunopräzipitation bestätigt werden. JNK1 phosphoryliert C/EBPα und erhöht dessen Halbwertszeit durch die Hemmung der Ubiquitinylierung. Dadurch wird die Stärke der Bindung von C/EBPα an die DNA und seine Fähigkeit zur Transaktivierung erhöht. Interessanterweise ist die Expression der JNK1-mRNA und die Kinaseaktivität von JNK1 bei AML-Patienten verglichen mit den entsprechenden Werten in mononukleären Zellen aus dem Knochenmark gesunder Kontrollpersonen erniedrigt. Dies weist auf einen möglichen Mechanismus zur funktionellen Inaktivierung von C/EBPα in bestimmten Subtypen der AML hin. 69 Zusammengefasst weisen unsere Daten darauf hin, dass die Aktivität von JNK1 für die Aktivierung von C/EBPα in myeloischen Zellen nötig ist, und dass ein Verlust der durch JNK1 regulierten Aktivität von C/EBPα einen möglichen Beitrag zur Leukämie-Entstehung leistet

E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation

AbstractTight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal–lysosomal routing and ubiquitin–proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin–proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp–Cullin–F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3β are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3β negatively regulates G-CSFR expression and its downstream signaling

Evaluation of Reirradiation in Locally Advanced Head and Neck Cancers: Toxicity and Early Clinical Outcomes

Objectives. Locoregional recurrence is the predominant pattern of treatment failure in advanced head and neck cancers. Reirradiation is a useful modality to treat inoperable head and neck cancer patients with recurrent disease. The aim of the present study was to analyze the treatment toxicity and early clinical outcomes in patients undergoing reirradiation. Methods. Twenty patients of head and neck cancers with recurrences or second cancers were evaluated. Reirradiation was done using simultaneous integrated boost volumetric modulated arc therapy (SIB VMAT), intensity modulated radiotherapy (IMRT), or conventional radiotherapy using 6MV photons. Dose prescription ranged from 30 to 60 Gy in conventional fractionation. Results. Seventeen males and three females were evaluated in this analysis. The median age of patients under study was 56.5 years. At time of analysis 8 patients (40%) had a complete response, 7 patients (35%) had progressive disease, and 25% had partial response or stable disease. Grade III-IV mucositis, dermatitis, xerostomia, dysphagia, and trismus were seen in 20%, 20%, 50%, 35%, and 45% patients, respectively, during retreatment. Patients receiving a radiotherapy dose less than 45 Gy showed a higher incidence of progressive disease (p=0.01). The median disease-free survival for patients receiving reirradiation dose of ≥46 Gy was 19±3.3 months (median ± S Error) compared to 8±2.61 months for those with a dose prescription less than 45 Gy (p=0.03). At 18-month follow-up 26% of patients undergoing reirradiation were disease-free. Conclusions. Our results show improved tumor control using a prescription of doses ≥46 Gy in retreatment setting

Population differentiation of Southern Indian male lineages correlates with agricultural expansions predating the caste system

Christina J. Adler, Alan Cooper, Clio S.I. Der Sarkissian and Wolfgang Haak are contributors to the Genographic ConsortiumPrevious studies that pooled Indian populations from a wide variety of geographical locations, have obtained contradictory conclusions about the processes of the establishment of the Varna caste system and its genetic impact on the origins and demographic histories of Indian populations. To further investigate these questions we took advantage that both Y chromosome and caste designation are paternally inherited, and genotyped 1,680 Y chromosomes representing 12 tribal and 19 non-tribal (caste) endogamous populations from the predominantly Dravidian-speaking Tamil Nadu state in the southernmost part of India. Tribes and castes were both characterized by an overwhelming proportion of putatively Indian autochthonous Y-chromosomal haplogroups (H-M69, F-M89, R1a1-M17, L1-M27, R2-M124, and C5-M356; 81% combined) with a shared genetic heritage dating back to the late Pleistocene (10–30 Kya), suggesting that more recent Holocene migrations from western Eurasia contributed, <20% of the male lineages. We found strong evidence for genetic structure, associated primarily with the current mode of subsistence. Coalescence analysis suggested that the social stratification was established 4–6 Kya and there was little admixture during the last 3 Kya, implying a minimal genetic impact of the Varna(caste) system from the historically-documented Brahmin migrations into the area. In contrast, the overall Y-chromosomal patterns, the time depth of population diversifications and the period of differentiation were best explained by the emergence of agricultural technology in South Asia. These results highlight the utility of detailed local genetic studies within India, without prior assumptions about the importance of Varna rank status for population grouping, to obtain new insights into the relative influences of past demographic events for the population structure of the whole of modern India.GaneshPrasad ArunKumar, David F. Soria-Hernanz, Valampuri John Kavitha, Varatharajan Santhakumari Arun, Adhikarla Syama, Kumaran Samy Ashokan, Kavandanpatti Thangaraj Gandhirajan, Koothapuli Vijayakumar, Muthuswamy Narayanan, Mariakuttikan Jayalakshmi, Janet S. Ziegle, Ajay K. Royyuru, Laxmi Parida, R. Spencer Wells, Colin Renfrew, Theodore G. Schurr, Chris Tyler Smith, Daniel E. Platt, Ramasamy Pitchappan, The Genographic Consortiu

In vivo assessment of dichlorvos induced histological and biochemical impairments coupled with expression of p53 responsive apoptotic genes in the liver and kidney of fish, Channa punctatus (Bloch, 1793)

Sub-lethal exposure of dichlorvos induces oxidative stress, consequent genetic instability and apoptosis coupled with impairments in biochemical, histopathological and transcription of genes in Channa punctatus. Exposure of 5% (0.041 mg/L; E2) and 10% (0.082 mg/L; E3) of 96 h-LC50 of dichlorvos significantly (p < 0.05) elevated the reactive oxygen species (ROS) generation and activities of SOD and CAT, as compared to control (E1) after 30 d. The maximum reduction in reduced glutathione (GSH) was recorded in the liver (18.53 ± 0.81 μg/mg of protein) and kidney (19.32 ± 0.97 μg/mg of protein); while the total protein contents were also found reduced, 278.38 ± 8.40 μg/mL (liver) and 248.44 ± 7.28 μg/mL (kidney), after 30 days in E3, in comparison to respective controls. Further, significant (p < 0.05) induction in micronuclei (MN) and apoptotic cells (AC), in a dose- and exposure-based manner were also recorded. Moreover, a significant (p < 0.05) up-regulation of p53 (2.51-fold in liver), bax (2.03-fold in liver; 1.99-fold in kidney) and casp3a (2.26-fold in liver; 2.10-fold in kidney) together with an elevated expression of cat (1.73-fold in liver; 1.12-fold in kidney), p53 (1.27-fold in kidney) and apaf-1 (1.72-fold in liver) in fish exposed to higher dose of dichlorvos for 30 d evidently reflects geno-toxicological potential of referenced pesticide. Disturbed biochemical and molecular parameters evince that the fish experienced oxidative stress as is further supported by prominent pathological observations in liver and kidney. Findings are, thus, helpful in organ-specific molecular scanning against aquatic toxicants like dichlorvos. [Display omitted] •Chronically, dichlorvos exposure significantly (p < 0.05) elicits ROS, SOD and CAT activities in fish.•GSH and total protein contents decreased significantly (p < 0.05) in liver and kidney.•Micronuclei (MN) and apoptotic cells (AC) frequencies increased significantly (p < 0.05) in fish erythrocytes.•Differential transcriptional regulations of cat, p53, bax, apaf-1 and casp3a genes exhibit genomic instability and apoptosis.•The prominent histopathological alterations were observed in liver and kidney

A protective study of curcumin associated with Cr6+ induced oxidative stress, genetic damage, transcription of genes related to apoptosis and histopathology of fish, Channa punctatus (Bloch, 1793)

[Display omitted] •Curcumin ameliorates Cr6+ induced hepatoxicity in Channa punctatus.•Curcumin reduces Cr6+ induced oxidative stress by decreasing the activities of SOD, CAT, and GR.•Curcumin counteracts against Cr6+ induced genotoxicity.•Curcumin significantly (p < 0.05) recovered the changes in transcripts of genes. Ameliorative potential of curcumin against Cr6+-induced eco-toxicological manifestations was assessed in liver of exposed Channa punctatus (Actinopterygii) in six groups for 45 d; Group I as control. Group II with 3 mg/L of curcumin; group III with 7.89 mg/L of Cr6+. Groups IV, V and VI were simultaneously co-exposed with 7.89 mg/L of Cr6+ and three different curcumin concentrations, 1, 2, and 3 mg/L, respectively. In group III, SOD-CAT, GR significantly (p < 0.05) increased; decreased GSH level; elevated MN and AC frequencies; and a significant (p < 0.05) up-regulation of cat (2.72-fold), p53 (1.73-fold), bax (1.33-fold) and apaf-1 (2.13-fold) together with a significant (p < 0.05) down-regulation of bcl-2 (0.51-fold). Co-exposure significantly (p < 0.05) brought down activities of SOD-CAT, GR, raised GSH, decreased micronuclei and apoptotic frequencies along with recovery of histopathological anomalies in liver. This study establishes the protective role of curcumin against Cr6+-induced hepatotoxicity in fish

Biomonitoring of Heavy Metals in River Ganga Water, Sediments, Plant, and Fishes of Different Trophic Levels

In this study, the pattern of metals concentration in water, sediment, plants, and three edible fish species (Channa striata, Labeo rohita, and Catla catla) of different trophic levels, captured from Jajmau (Kanpur), an important fishery area of river Ganga in Uttar Pradesh, India was examined. The heavy metals, Ni, Pb, Fe, Cu, Zn, Cd, Cr, and Co, were estimated in the liver, kidney, muscles, and gill tissues of abovesaid species of fish. The highest metal concentration was reported in the bottom feeder fish as compared with the column and surface feeders. The result obtained after analysis of water sample reflects the order of occurrence of heavy metals as Fe > Cr > Pb > Ni > Cd > Zn > Cu > Co. Sediments analysis indicates high concentration of Fe and Cr, making the entire environment from top to bottom, stressful. Target hazard quotient (THQ) and hazard index (HI) of the three species suggest a potential risk to the health of consumers, the humans. Thus, it is inevitable that the river Ganga should be closely monitored to safeguard human health. Graphical Abstract

An in vivo analysis of Cr6+ induced biochemical, genotoxicological and transcriptional profiling of genes related to oxidative stress, DNA damage and apoptosis in liver of fish, Channa punctatus (Bloch, 1793)

•Bioaccumulation of Cr6+ induces oxidative stress in liver of C. punctatus exposed to 5 and 10% of 96 h-LC50 of CrO3.•Oxidative stress was assessed to determine SOD and CAT activities in liver of fish exposed chronically.•A significant (p < 0.05) induction of MN in fish erythrocytes suggests that Cr6+ is genotoxic.•Differential expression of target genes related to oxidative stress, DNA damage and apoptosis in fish.•Molecular markers can be efficiently utilized for biomonitoring and conservation studies. Present study was designed to assess the hexavalent chromium (Cr6+) mediated oxidative stress that induces DNA damage and apoptosis in adult fish, Channa punctatus (35 ± 3.0 g; 14.5 ± 1.0 cm; Actinopterygii). Fishes were maintained in three groups for 15, 30 and 45 d of exposure periods. They were treated with 5% (Group T1) and 10% (Group T2) of 96 h-LC50 of chromium trioxide (Cr6+). Controls were run for the similar duration. A significant (p < 0.05) increment in the activities of antioxidant enzymes, SOD and CAT in liver tissues of the exposed fish evinces the persistence of oxidative stress. A significant (p < 0.05) increase in induction of micronuclei (MN) coupled with transcriptional responses of target genes related to antioxidant enzymes, DNA damage and apoptosis (sod, cat, gsr, nox-1, p53, bax, bcl-2, apaf-1 and casp3a) establishes the impact of oxidative stress due to in vivo, Cr6+ accumulation in liver as compared to control (0 mg/L), in a dose and exposure-dependent manner. Initially, the increased level of reactive oxygen species (ROS) in liver coincided with that of enhanced mRNA expression of antioxidant enzymes, sod, cat, gsr and nox-1 but, later, the overproduction of ROS, after 45 d of exposure of Cr6+, resulted in a significant (p < 0.05) up-regulation of p53. Our findings also unveil that the up-regulation of bax, apaf-1 and casp3a and down-regulation of bcl-2 are associated with Cr6+-induced oxidative stress mediated-apoptosis in liver of test fish. Aforesaid molecular markers can, thus, be efficiently utilized for bio-monitoring of aquatic regimes and conservation of fish biodiversity

Phorate induced oxidative stress, DNA damage and differential expression of p53, apaf-1 and cat genes in fish, Channa punctatus (Bloch, 1793)

The present study was conducted to assess the in-vivo activities of certain molecular biomarkers under the impact of phorate exposure. Fish, Channa punctatus (35 ± 3.0 g; 14.5 ± 1.0 cm; Actinopterygii) were subjected to semi-static conditions having 5% (0.0375 mg/L for T1 group) and 10% of 96 h-LC50 (0.075 mg/L for T2 group) of phorate exposure for 15 and 30 d. The oxidative stress was assessed in terms of superoxide dismutase (SOD) and catalase (CAT) activities. DNA damage was measured as induction of micronuclei (MN) and consequent differential expression of apoptotic genes-tumor suppressor (p53), apoptotic peptidase activating factor-1 (apaf-1) and catalase (cat) in liver and kidney, two major sites of biotransformation in fish, were quantified. Our findings reveal significant (p < 0.001) augmentations in SOD and CAT activities of liver and kidney tissues. MN frequency in erythrocytes of fish also increases significantly (p < 0.05) in a dose- and time-dependent manner. The mRNA level of p53 increased significantly (p < 0.05) in liver at 10% of 96 h-LC50 of phorate exposure after 30 d suggesting generation of stress due to accumulation of reactive oxygen species (ROS). Eventually, these findings decipher the dual role of ROS in generating genotoxicity as is evident by micronuclei induction and differential regulation of p53, apaf-1 and cat genes during the phorate induced DNA damage and apoptosis in test fish. The experimental inferences drawn on the basis of activities of aforesaid biomarkers shall be helpful in elucidating the possible causes of apoptosis under stressful conditions. Further, this study finds ample application in biomonitoring of phorate polluted aquatic ecosystem. •Sub-lethal chronic exposure of phorate induces oxidative stress in fish.•SOD and CAT activities in liver and kidney tissues increased significantly (p < 0.001).•MN frequency in fish erythrocytes increases significantly (p < 0.05) in a dose and time-dependent manner.•Differential expression of cat, p53 and apaf-1 genes evinces DNA damage and apoptosis.•cat, p53 and apaf-1 genes can be used as sensitive biomarkers for monitoring of freshwater pollution