Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use. © 2011 John Wiley & Sons A/S.

Validation of the interRAI Cognitive Performance Scale against independent clinical diagnosis and the Mini-Mental State Examination in older hospitalized patients

Objective To compare the diagnostic accuracy of the interRAI Acute Care (AC) Cognitive Performance Scale (CPS2) and the Mini-Mental State Examination (MMSE), against independent clinical diagnosis for detecting dementia in older hospitalized patients. Design, Setting, and Participants The study was part of a prospective observational cohort study of patients aged ≥70 years admitted to four acute hospitals in Queensland, Australia, between 2008 and 2010. Recruitment was consecutive and patients expected to remain in hospital for ≥48 hours were eligible to participate. Data for 462 patients were available for this study. Measurements Trained research nurses completed comprehensive geriatric assessments and administered the interRAI AC and MMSE to patients. Two physicians independently reviewed patients’ medical records and assessments to establish the diagnosis of dementia. Indicators of diagnostic accuracy included sensitivity, specificity, predictive values, likelihood ratios and areas under receiver (AUC) operating characteristic curves. Results 85 patients (18.4%) were considered to have dementia according to independent clinical diagnosis. The sensitivity of the CPS2 [0.68 (95%CI: 0.58–0.77)] was not statistically different to the MMSE [0.75 (0.64–0.83)] in predicting physician diagnosed dementia. The AUCs for the 2 instruments were also not statistically different: CPS2 AUC = 0.83 (95%CI: 0.78–0.89) and MMSE AUC = 0.87 (95%CI: 0.83–0.91), while the CPS2 demonstrated higher specificity [0.92 95%CI: 0.89–0.95)] than the MMSE [0.82 (0.77–0.85)]. Agreement between the CPS2 and clinical diagnosis was substantial (87.4%; κ=0.61). Conclusion The CPS2 appears to be a reliable screening tool for assessing cognitive impairment in acutely unwell older hospitalized patients. These findings add to the growing body of evidence supporting the utility of the interRAI AC, within which the CPS2 is embedded. The interRAI AC offers the advantage of being able to accurately screen for both dementia and delirium without the need to use additional assessments, thus increasing assessment efficiency.

Angiogenesis is an independent prognostic factor in malignant mesothelioma

Angiogenesis is essential for tumour growth beyond 1 to 2 mm in diameter. The clinical relevance of angiogenesis, as assessed by microvessel density (MVD), is unclear in malignant mesothelioma (MM). Immunohistochemistry was performed on 104 archival, paraffin-embedded, surgically resected MM samples with an anti-CD34 monoclonal antibody, using the Streptavidin-biotin complex immunoperoxidase technique. 93 cases were suitable for microvessel quantification. MVD was obtained from 3 intratumoural hotspots, using a Chalkley eyepiece graticule at × 250 power. MVD was correlated with survival by Kaplan-Meier and log-rank analysis. A stepwise, multivariate Cox model was used to compare MVD with known prognostic factors and the EORTC and CALGB prognostic scoring systems. Overall median survival from the date of diagnosis was 5.0 months. Increasing MVD was a poor prognostic factor in univariate analysis (P = 0.02). Independent indicators of poor prognosis in multivariate analysis were non-epithelial cell type (P = 0.002), performance status > 0 (P = 0.003) and increasing MVD (P = 0.01). In multivariate Cox analysis, MVD contributed independently to the EORTC (P = 0.006), but not to the CALGB (P = 0.1), prognostic groups. Angiogenesis, as assessed by MVD, is a poor prognostic factor in MM, independent of other clinicopathological variables and the EORTC prognostic scoring system. Further work is required to assess the prognostic importance of angiogenic regulatory factors in this disease. © 2001 Cancer Research Campaign.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

Chronic immune activation and inflammation as the cause of malignancy

Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers. © 2001 Cancer Research Campaign http://www.bjcancer.com.

Phospho-Akt expression is associated with a favorable outcome in non-small cell lung cancer

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037). These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC. © 2005 American Association for Cancer Research.

Tumour necrosis is an independent prognostic marker in non-small cell lung cancer : correlation with biological variables

Background Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. Methods In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. Results Reproducibility was attained in 36/40 cases, Kappa score=0.8 P<0.001. TN correlated with T-stage (P=0.001), platelet count (P=0.004) and p53 expression (P=0.031). Near significant associations of TN with N-stage (P=0.063) and MMP-9 expression (P=0.058) were seen. No association was found with angiogenesis (P=0.98). On univariate (P=0.0016) and multivariate analysis (P=0.023) TN was prognostic. Conclusion These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Pro-osteogenic topographical cues promote early activation of osteoprogenitor differentiation via enhanced TGFβ, Wnt, and Notch signaling

Objectives Titanium implant surfaces with modified topographies have improved osteogenic properties in vivo. However, the molecular mechanisms remain obscure. This study explored the signaling pathways responsible for the pro-osteogenic properties of micro-roughened (SLA) and chemically/nanostructurally (modSLA) modified titanium surfaces on human alveolar bone-derived osteoprogenitor cells (BCs) in vitro. Materials and methods The activation of stem cell signaling pathways (TGFβ/BMP, Wnt, FGF, Hedgehog, Notch) was investigated following early exposure (24 and 72 h) of BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture supplements. Results Key regulatory genes from the TGFβ/BMP (TGFBR2, BMPR2, BMPR1B, ACVR1B, SMAD1, SMAD5), Wnt (Wnt/β-catenin and Wnt/Ca2+) (FZD1, FZD3, FZD5, LRP5, NFATC1, NFATC2, NFATC4, PYGO2, LEF1) and Notch (NOTCH1, NOTCH2, NOTCH4, PSEN1, PSEN2, PSENEN) pathways were upregulated on the modified surfaces. These findings correlated with a higher expression of osteogenic markers bone sialoprotein (IBSP) and osteocalcin (BGLAP), and bone differentiation factors BMP2, BMP6, and GDF15, as observed on the modified surfaces. Conclusions These findings demonstrate that the activation of the pro-osteogenic cell signaling pathways by modSLA and SLA surfaces leads to enhanced osteogenic differentiation as evidenced after 7 and 14 days culture in osteogenic media and provides a mechanistic insight into the superior osseointegration on the modified surfaces observed in vivo.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer

RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract. © CSIRO 2008.

High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation

Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

On ‘Activation Workers' Perceptions’ : a reply to Dunn (1)

Andrew Dunn challenges us to include the perspectives of other policy actors in the debate about the merits and limits of activation policies that emphasise greater conditionality for those in receipt of benefits. In his provocative article he focuses on the views and experiences of people who work with unemployed people...

From Aussiewood Movies to Guerrilla Filmmaking: Independent Filmmaking and Contemporary Australian Cinema

Independent filmmaking within the context of Australian cinema is a multifaceted subject. In comparison to the United States, where production can be characterised as bifurcated between major studio production and so-called “indie” or independent production without the backing of the majors, since the 1970s and until recently the vast majority of Australian feature film production has been independent filmmaking. Like most so-called national cinemas, most Australian movies are supported by both direct and indirect public subvention administered by state and federal government funding bodies, and it could be argued that filmmakers are, to a certain degree, “dependent” on official mandates. As this chapter demonstrates national production slates are subjected to budget restraints and cut-backs, official cultural policies (for example pursuing international co-productions and local content quotas) and shifts in policy directions among others. Therefore, within the context of Australian cinema, feature film production operating outside the public funding system could be understood as “independent”. However, as is the case for most English-language national cinemas, independence has long been defined in terms of autonomy from Hollywood, and – as alluded to above – as Australia becomes more dependent upon international inputs into production, higher budget movies are becoming less independent from Hollywood. As such, this chapter argues that independence in Australian cinema can be viewed as having two poles: independence from direct government funding and independence from Hollywood studios. With a specific focus on industry and policy contexts, this chapter explores key issues that constitute independence for Australian cinema. In so doing it examines the production characteristics of four primary domains of contemporary independent filmmaking in Australia, namely: “Aussiewood” production; government-backed low-to-mid budget production; co-productions; and guerrilla filmmaking.

Neutrophil oxidative burst is primed by supernatant from stored red cells : implications for Transfusion-Related Acute Lung Injury (TRALI)

Aim/Background: Transfusion-related acute lung injury (TRALI) is a potentially fatal adverse transfusion reaction. It is hypothesised to occur via a two-insult mechanism: the recipient’s underlying co-morbidity in addition to the transfusion of blood products activate neutrophils in the lung resulting in damaged endothelium and capillary leakage. Neutrophil activation may occur by antibody or non-antibody related mechanisms, with the length of storage of cellular blood products implicated in the latter. This study investigated non-antibody mediated priming and/or activation of neutrophil oxidative burst. Methods: A cytochrome C reduction assay was used to assess priming and activation of neutrophil oxidative burst by pooled supernatant (SN) from day 1 (D1; n=75) and day 42 (D42; n=113) packed red blood cells (PRBC). Pooled PRBC-SN were assessed in parallel with PAF (priming), fMLP (activating), PAF + fMLP (priming + activating) and buffer only (negative) controls. Cytochrome C reduction was measured over 30min at 37oC (inclusive of 10min priming). Neutrophil activation by PRBC-SN was assessed cf. buffer only and neutrophil priming by PRBC-SN was assessed by co-incubation with fMLP cf. fMLP alone. One-way ANOVA; Newman-Keuls post-test; p<0.05; n=10 independent assays. Results: Neither D1- nor D42- PRBC-SN alone activated neutrophil oxidative burst. In addition, D1-PRBC-SN did not prime fMLP-activated neutrophil oxidative burst. D42-PRBC-SN did, however, prime neutrophils for subsequent activation of oxidative burst by fMLP, the magnitude of response being similar to PAF (a known neutrophil priming agonist). Conclusion: These findings are consistent with the two-insult mechanism of TRALI. Factors released into the SN during PRBC storage contributed to neutrophil priming synergistically with other neutrophil stimulating agonists. This implicates PRBC storage duration as a key factor contributing to non-immune neutrophil activation in the development of TRALI in patients with pre-disposing inflammatory conditions.

Controlling whole blood activation and resultant clot properties by carboxyl and alkyl functional groups on material surfaces : a possible therapeutic approach for enhancing bone healing

Most research virtually ignores the important role of a blood clot in supporting bone healing. In this study, we investigated the effects of surface functional groups carboxyl and alkyl on whole blood coagulation, complement activation and blood clot formation. We synthesised and tested a series of materials with different ratios of carboxyl (–COOH) and alkyl (–CH3, –CH2CH3 and –(CH2)3CH3) groups. We found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/– CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of coagulation activation. The pattern of complement activation was entirely similar to that of surface-induced coagulation. All material coated surfaces resulted in clots with thicker fibrin in a denser network at the clot/material interface and a significantly slower initial fibrinolysis when compared to uncoated glass surfaces. The amounts of platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-b (TGF-b1) released from an intact clot were higher than a lysed clot. The release of PDGF-AB was found to be correlated with the fibrin density. This study demonstrated that surface chemistry can significantly influence the activation of blood coagulation and complement system, resultant clot structure, susceptibility to fibrinolysis as well as release of growth factors, which are important factors determining the bone healing process.