Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation

Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST–XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at −80°C for at least 60 days. M30-Apoptosense™ plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at −80°C, while at 37°C it had a half-life of 80–100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis

Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatin's effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy

Inhibitors of apoptosis proteins in human cervical cancer

BACKGROUND: It has been shown that IAPs, in particular XIAP, survivin and c-IAP1, are overexpressed in several malignancies. In the present study we investigate the expression of c-IAP1, c-IAP2, XIAP and survivin and its isoforms in cervical cancer. METHODS: We used semiquantitative RT-PCR assays to analyze 41 cancer and 6 normal tissues. The study included 8 stage I cases; 16 stage II; 17 stageIII; and a control group of 6 samples of normal cervical squamous epithelial tissue. RESULTS: c-IAP2 and XIAP mRNA levels were similar among the samples, cervical tumors had lower c-IAP1 mRNA levels. Unexpectedly, a clear positive association was found between low levels of XIAP and disease relapse. A log-rank test showed a significant inverse association (p = 0.02) between XIAP expression and tumor aggressiveness, as indicated by disease relapse rates. There were no statistically significant differences in the presence or expression levels of c-IAP1 and c-IAP2 among any of the clinical variables studied. Survivin and its isoforms were undetectable in normal cervical tissues, in contrast with the clear upregulation observed in cancer samples. We found no association between survivin expression and age, clinical stage, histology or menopausal state. Nevertheless, we found that adenocarcinoma tumors expressed higher levels of survivin 2B and DeltaEx3 (p = 0.001 and p = 0.04 respectively, by Kruskal-Wallis). A multivariate Cox's partial likelihood-based analysis showed that only FIGO stage was an independent predictor of outcome. CONCLUSION: There are no differences in the expression of c-IAP2 and XIAP between normal vs. cancer samples, but XIAP expression correlate in cervical cancer with relapse of this disease in the patients. Otherwise, c-IAP1 was downregulated in the cervical cancer samples. The expression of survivin was upregulated in the patients with cervical cancer. We have found that adenocarcinoma presented higher levels of survivin isoforms 2B and DeltaEx3

Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity

Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection

Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes

A Role for Drosophila dFoxO and dFoxO 5′UTR Internal Ribosomal Entry Sites during Fasting

One way animals may cope with nutrient deprivation is to broadly repress translation by inhibiting 5′-cap initiation. However, under these conditions specific proteins remain essential to survival during fasting. Such peptides may be translated through initiation at 5′UTR Internal Ribosome Entry Sites (IRES). Here we show that the Drosophila melanogaster Forkhead box type O (dFoxO) transcription factor is required for adult survival during fasting, and that the 5′UTR of dfoxO has the ability to initiate IRES-mediated translation in cell culture. Previous work has shown that insulin negatively regulates dFoxO through AKT-mediated phosphorylation while dFoxO itself induces transcription of the insulin receptor dInR, which also harbors IRES. Here we report that IRES-mediated translation of both dFoxO and dInR is activated in fasted Drosophila S2 cells at a time when cap-dependent translation is reduced. IRES mediated translation of dFoxO and dInR may be essential to ensure function and sensitivity of the insulin signaling pathway during fasting

Activation of Type I and III Interferon Signalling Pathways Occurs in Lung Epithelial Cells Infected with Low Pathogenic Avian Influenza Viruses

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture

X-linked inhibitor of apoptosis positive nuclear labeling: a new independent prognostic biomarker of breast invasive ductal carcinoma

<p>Abstract</p> <p>Background</p> <p>It's well recognized that X-linked inhibitor of apoptosis (XIAP) was the most potent caspase inhibitor and second mitochondria-derived activator of caspase (Smac) was the antagonist of XIAP. Experiments in vitro identified that down regulation of XIAP expression or applying Smac mimics could sensitize breast cancer cells to chemotherapeutics and promote apoptosis. However, expression status and biologic or prognostic significance of XIAP/Smac in breast invasive ductal carcinoma (IDC) were not clear. The present study aimed to investigate relationship among expression status of XIAP/Smac, apoptosis index (AI), clinicopathologic parameters and prognosis in IDC.</p> <p>Methods</p> <p>Immunohistochemistry and TUNEL experiment were performed to detect expression of XIAP, Smac, ER, PR, HER2 and AI in 102 cases of paraffin-embedded IDC samples respectively. Expression of XIAP/Smac were also detected in limited 8 cases of fresh IDC specimens with Western blot.</p> <p>Results</p> <p>Positive ratio and immunoscore of XIAP was markedly higher than Smac in IDC (<it>P </it>< 0.0001). It was noteworthy that 44 cases of IDC were positive in nuclear for XIAP, but none was for Smac. Expression status of Smac was more prevalent in HER2 positive group than negative group (<it>P </it>< 0.0001) and AI was positively correlated with HER2 protein expression (r_s= 0.265, <it>P </it>= 0.017). The present study first revealed that XIAP positive nuclear labeling (XIAP-N), but not cytoplasmic staining (XIAP-C), was the apoptotic marker correlated significantly with patients' shortened overall survival (<it>P </it>= 0.039). Survival analysis demonstrated that XIAP-N was a new independent prognostic factor except for patient age and lymph node status.</p> <p>Conclusion</p> <p>Disturbed balance of expression between XIAP and Smac probably contributed to carcinogenesis and XIAP positive nuclear labeling was a new independent prognostic biomarker of breast IDC.</p

Ontogenetic Development of Weberian Ossicles and Hearing Abilities in the African Bullhead Catfish

BACKGROUND: The weberian apparatus of otophysine fishes facilitates sound transmission from the swimbladder to the inner ear to increase hearing sensitivity. It has been of great interest to biologists since the 19(th) century. No studies, however, are available on the development of the weberian ossicles and its effect on the development of hearing in catfishes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the development of the weberian apparatus and auditory sensitivity in the catfish Lophiobagrus cyclurus. Specimens from 11.3 mm to 85.5 mm in standard length were studied. Morphology was assessed using sectioning, histology, and X-ray computed tomography, along with 3D reconstruction. Hearing thresholds were measured utilizing the auditory evoked potentials recording technique. Weberian ossicles and interossicular ligaments were fully developed in all stages investigated except in the smallest size group. In the smallest catfish, the intercalarium and the interossicular ligaments were still missing and the tripus was not yet fully developed. Smallest juveniles revealed lowest auditory sensitivity and were unable to detect frequencies higher than 2 or 3 kHz; sensitivity increased in larger specimens by up to 40 dB, and frequency detection up to 6 kHz. In the size groups capable of perceiving frequencies up to 6 kHz, larger individuals had better hearing abilities at low frequencies (0.05-2 kHz), whereas smaller individuals showed better hearing at the highest frequencies (4-6 kHz). CONCLUSIONS/SIGNIFICANCE: Our data indicate that the ability of otophysine fish to detect sounds at low levels and high frequencies largely depends on the development of the weberian apparatus. A significant increase in auditory sensitivity was observed as soon as all weberian ossicles and interossicular ligaments are present and the chain for transmitting sounds from the swimbladder to the inner ear is complete. This contrasts with findings in another otophysine, the zebrafish, where no threshold changes have been observed