Glioblastoma multiforme is the most aggressive form of primary brain tumor and remains largely incurable, in large part, due to its highly invasive nature. The phosphoinositide (PI) 3-kinase pathway ...is often constitutively active in these tumors due to activating mutations in the epidermal growth factor receptor, or deletion/loss of function of the tumor suppressor PTEN. Protein kinase C type iota (PKC iota), a member of the atypical protein kinase C family, is activated by the PI 3-kinase pathway and is an important downstream mediator. Here, we have assessed the role of PKC iota in glioblastoma cell invasion. Depletion of PKC iota with RNA interference caused an increase in actin stress fibers and a decrease in cell motility and invasion. Gene expression microarray analysis of U87MG cells showed that PKC iota repressed expression of mRNA for RhoB, which has previously been shown to have a role in actin stress fiber formation. Western blot analysis showed that both PKC iota depletion and pharmacological inhibition of PKC iota caused an increase in the protein levels of RhoB, as did inhibition of PI 3-kinase. Expression of RhoB from a constitutive promoter caused changes in actin stress fibers and cell invasion that were similar to those seen with PKC iota depletion. These data show that PKC iota, activated as a consequence of aberrant upstream PI 3-kinase signaling, mediates glioblastoma cell motility and invasion, and that repression of RhoB is key downstream event in PKC iota signaling leading to enhanced cell motility. In addition, constitutive expression of RhoB repressed PKC iota activity, as assessed by its phosphorylation status on Thr555. PKC iota and RhoB are, therefore, mutually antagonistic, potentially creating a sensitive switch between invasive and non-invasive phenotypes.
To evaluate the psychometric properties of the 12-item Hip disability and Osteoarthritis Outcome Score (HOOS-12) and Knee injury and Osteoarthritis Outcome Score (KOOS-12) for use in evaluating ...outcomes after joint replacement for osteoarthritis.
Patient-reported outcomes data collected by the Australian Orthopaedic Association National Joint Replacement Registry were used for this analysis. HOOS-12 and KOOS-12 domain (pain, function, quality of life) and summary impact data were available. The Oxford Hip Score (OHS), Oxford Knee Score (OKS) and EQ-5D-5L were used as comparators. Instruments were administered pre-operatively and at 6 months post-operatively. Internal consistency reliability, floor and ceiling effects, convergent validity, known groups validity, and responsiveness were evaluated using standard psychometric techniques.
Baseline HOOS-12 and KOOS-12 data were available for 3,023 patients undergoing primary total hip replacement and 4,010 patients undergoing primary total knee replacement. At baseline, high internal consistency was demonstrated for all domains and summary scores (Cronbach's alpha: HOOS-12 = 0.81–0.93; KOOS-12 = 0.82–0.92). Post-operative ceiling effects (>15% of patients scoring the best possible score) were identified for the HOOS-12 pain (46%), function (39%) and quality of life domains (26%) and summary score (17%), and for the KOOS-12 pain (21%) and function domains (18%). The HOOS-12 and KOOS-12 could differentiate between two known groups (lowest/highest OHS or OKS quartiles post-operatively; p < 0.001) and were highly responsive to change (effect sizes for HOOS-12: 2.20–2.83; KOOS-12: 1.82–2.35).
The HOOS-12 and KOOS-12 have good psychometric properties for capturing joint replacement outcomes including excellent responsiveness, although ceiling effects may limit monitoring of post-operative improvement.
To evaluate the structural validity of the 12-item Hip disability and Osteoarthritis Outcome Score (HOOS-12) and 12-item Knee injury and Osteoarthritis Outcome Score (KOOS-12) using Rasch analysis ...and consider psychometric implications for research and clinical use.
Individual-level HOOS-12 and KOOS-12 data from the Australian Orthopaedic Association National Joint Replacement Registry, collected before and after primary total hip and knee replacement, were used for this analysis. Using the Rasch analytic approach, overall model fit and item fit were examined, together with potential reasons for misfit including response threshold ordering, differential item functioning, internal consistency, unidimensionality and item targeting.
Overall misfit to the Rasch model was evident for both instruments. A degree of item misfit was also observed, although most items demonstrated logical sequencing of response options. Only two items (hip/knee pain frequency and awareness of hip/knee problems) displayed disordered response thresholds. The pain, function, and quality of life domains of the HOOS-12 and KOOS-12 demonstrated excellent internal consistency reliability (person separation index: 0.80–0.93) and unidimensionality. A mismatch between item difficulty and person ability scores at the highest end of the HOOS-12 and KOOS-12 scales contributed to post-operative ceiling effects (mean logit for HOOS-12: 3.57; KOOS-12: 2.58; ≈0 indicates well-targeted scale).
We found evidence to support the structural validity of the three HOOS-12 and KOOS-12 domains for evaluating joint replacement outcomes. However, there may be missing content in both instruments particularly for high-functioning patients. Minor refinement of some response options may be warranted to improve item performance.
Senescence is an irreversible growth arrest phenotype adopted by cells that has a key role in protecting organisms from cancer. There is now considerable interest in therapeutic strategies that ...reactivate this process to control the growth of cancer cells. Protein kinase-Cι (PKCι) is a member of the atypical PKC family and an important downstream mediator in the phosphoinositide-3-kinase (PI-3-kinase) pathway. PKCι expression was found to be upregulated in a subset of breast cancers and breast cancer cell lines. Activation of the PI-3-kinase pathway by introduction of mutant, oncogenic PIK3CA into breast mammary epithelial cells increased both the expression and activation of PKCι. In breast cancer cells lines overexpressing PKCι, depletion of PKCι increased the number of senescent cells, as assessed by senescence-associated β-galactosidase, morphology and bromodeoxyuridine incorporation. This phenomenon was not restricted to breast cancer cells, as it was also seen in glioblastoma cells in which PKCι is activated by loss of PTEN. Senescence occurred in the absence of a detectable DNA-damage response, was dependent on p21 and was enhanced by the aurora kinase inhibitor VX-680, suggesting that senescence is triggered by defects in mitosis. Depletion of PKCι had no effect on senescence in normal mammary epithelial cell lines. We conclude that PKCι is overexpressed in a subset of cancers where it functions to suppress premature senescence. This function appears to be restricted to cancer cells and inhibition of PKCι may therefore be an effective way to selectively activate premature senescence in cancer cells.
We have carried out a search for radio emission at 820 MHz from six X-ray dim isolated neutron stars (XDINSs) with the Robert C. Byrd Green Bank Radio Telescope. No transient or pulsed emission was ...found using fast folding, fast Fourier transform, and single-pulse searches. The corresponding flux limits are about 0.01 mJy for pulsed emission, depending on the integration time for the particular source and assuming a duty cycle of 2%, and 20 mJy for single dispersed pulses. These are the most sensitive limits to date on radio emission from XDINSs. There is no evidence for isolated radio pulses, as seen in a class of neutron stars known as rotating radio transients. Our results imply that either the radio luminosities of these objects are lower than those of any known radio pulsars, or they could simply be long-period nearby radio pulsars with high magnetic fields beaming away from the Earth. To test the latter possibility, we would need around 40 similar sources to provide a 1 Delta *s probability of at least one of them beaming toward us. We also give a detailed description of our implementation of the Fast Folding Algorithm.
We present radio transient search algorithms, results, and statistics from the ongoing Arecibo Pulsar ALFA (PALFA) survey of the Galactic plane. We have discovered seven objects through a search for ...isolated dispersed pulses. All of these objects are Galactic and have measured periods between 0.4 and 4.7 s. One of the new discoveries has a duty cycle of 0.01%, smaller than that of any other radio pulsar. We discuss the impact of selection effects on the detectability and classification of intermittent sources, and compare the efficiencies of periodicity and single-pulse (SP) searches for various pulsar classes. For some cases we find that the apparent intermittency is likely to be caused by off-axis detection or a short time window that selects only a few bright pulses and favors detection with our SP algorithm. In other cases, the intermittency appears to be intrinsic to the source. No transients were found with DMs large enough to require that they originate from sources outside our Galaxy. Accounting for the on-axis gain of the ALFA system, as well as the low gain but large solid-angle coverage of far-out sidelobes, we use the results of the survey so far to place limits on the amplitudes and event rates of transients of arbitrary origin.
The epidermal growth factor (EGF) receptor is overexpressed in many cancers, and is under intensive investigation as a target for cancer therapy. Cancer cells have also been shown to express mutated ...EGF receptors; these are potentially highly specific targets for cancer therapeutics, as they have not been detected in any normal adult tissues. The most common of these mutant EGF receptors, EGFRvIII, is one in which amino acids 6 - 273 of the extracellular domain are deleted. This specific mutation is common in glioblastoma and in several other types of cancer, and has been shown to promote aggressive growth of tumors in vivo. The loss of part of the extracellular domain results in a receptor that has constitutive tyrosine kinase activity. Current evidence suggests that EGFRvIII has altered signalling properties compared to normal EGF receptor. The mutation in EGFRvIII also creates a new, cancer cell-specific epitope. This epitope is extracellular and therefore represents a very promising target for antibody-directed therapeutics. This review covers our current understanding of the properties of EGFRvIII, and recent developments in the characterization and therapeutic application of EGFRvIII-specific antibodies.
Glioblastoma multiforme is an aggressive form of brain cancer that responds poorly to chemotherapy and is generally incurable. The basis for the poor response of this cancer to chemotherapy is not ...well understood. The atypical protein kinases C (PKCι and PKCζ) have previously been implicated in leukaemia cell chemoresistance. To assess the role of atypical PKC in glioblastoma cell chemoresistance, RNA interference was used to deplete human glioblastoma cells of PKCι. Transfection of cells with either of two different RNA duplexes specific for PKCι caused a partial sensitisation to cell death induced by the chemotherapy agent cisplatin. To screen for possible mechanisms for PKCι-mediated chemoresistance, microarray analysis of gene expression was performed on RNA from glioblastoma cells that were either untreated or depleted of PKCι. This identified sets of genes that were regulated either positively or negatively by PKCι. Within the set of genes that were negatively regulated by PKCι, the function of the gene coding for GMFβ, an enhancer of p38 mitogen-activated protein kinase (MAP kinase) signaling, was investigated further, as the p38 MAP kinase pathway has been previously identified as a key mediator of cisplatin cytotoxicity. The expression of both GMFβ mRNA and protein increased upon PKCι depletion, and this was accompanied by an increase in cisplatin-activated p38 MAP kinase signaling. Transient overexpression of GMFβ increased cisplatin-activated p38 MAP kinase signaling and also sensitised cells to cisplatin cytotoxicity. The increase in cisplatin cytotoxicity seen with PKCι depletion was blocked by the p38 MAP kinase inhibitor SKF86002. These data show that PKCι can confer partial resistance to cisplatin in glioblastoma cells by suppressing GMFβ-mediated enhancement of p38 MAP kinase signaling.
The epidermal growth factor receptor (EGFR) is a key regulator of growth, differentiation, and survival of epithelial cancers. In a small subset of tumors, the presence of activating mutations within ...the ATP binding site confers increased susceptibility to gefitinib, a potent tyrosine kinase inhibitor of EGFR. Agents that can inhibit EGFR function through different mechanisms may enhance gefitinib activity in patients lacking these mutations. Mevalonate metabolites play significant roles in the function of the EGFR; therefore, mevalonate pathway inhibitors may potentiate EGFR-targeted therapies.
In this study, we evaluated the effect of lovastatin on EGFR function and on gefitinib activity. Effects on EGFR function were analyzed by Western blot analysis using phosphospecific antibodies to EGFR, AKT, and extracellular signal-regulated kinase. Cytotoxic effects of lovastatin and/or gefitinib were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry.
Lovastatin treatment inhibited EGF-induced EGFR autophosphorylation by 24 hours that was reversed by the coadministration of mevalonate. Combining lovastatin and gefitinib treatments showed enhanced inhibition of AKT activation by EGF in SCC9 cells. The combination of 10 mumol/L lovastatin and 10 mumol/L gefitinib treatments showed cooperative cytotoxicity in all 8 squamous cell carcinomas, 4 of 4 non-small cell lung carcinoma and 4 of 4 colon carcinoma cell lines tested. Isobologram and flow cytometric analyses of three representative cell lines with wild-type EGFR ATP binding sites confirmed that this combination was synergistic inducing a potent apoptotic response.
Taken together, these results show that targeting the mevalonate pathway can inhibit EGFR function. They also suggest the potential utility of combining these clinically relevant therapeutic approaches.
PDF
