Hypoxia-inducible factor-1α in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathways

Hypoxia-inducible factor (HIF)-1α is the regulatory subunit of HIF-1 that is stabilized under hypoxic conditions. Under different circumstances, HIF-1α may promote both tumorigenesis and apoptosis. There is conflicting data on the importance of HIF-1α as a prognostic factor. This study evaluated HIF-1α expression in 172 consecutive patients with stage I-IIIA non small cell lung cancer (NSCLC) using standard immunohistochemical techniques. The extent of HIF-1α nuclear immunostaining was determined using light microscopy and the results were analyzed using the median (5%) as a low cut-point and 60% as a high positive cut-point. Using the low cut-point, positive associations were found with epidermal growth factor receptor (EGFR; p = 0.01), matrix metalloproteinase (MMP)-9 (p = 0.003), membranous (p < 0.001) and perinuclear (p = 0.004) carbonic anhydrase (CA) IX, pS3 (p = 0.008), T-stage (p = 0.042), tumor necrosis (TN; p < 0.001) and squamous histology (p < 0.001). No significant association was found with Bcl-2 or either N- or overall TMN stage or prognosis. When the high positive cut-point was used, HIF-1α was associated with a poor prognosis (p = 0.034). In conclusion, the associations with EGFR, MMP-9, p53 and CA IX suggest that these factors may either regulate or be regulated by HIF-1α. The association with TN and squamous-type histology, which is relatively more necrotic than other NSCLC types, reflects the role of hypoxia in the regulation of HIF-1α. The prognostic data may reflect a change in the behavior of HIF-1α in increasingly hypoxic environments. © 2004 Wiley-Liss, Inc.

Interactions between hypoxia and epidermal growth factor receptor in non-small-cell-lung cancer

Tumor hypoxia has been recognized to confer resistance to anticancer therapy since the early 20th century. More recently, its fundamental role in tumorigenesis has been established. Hypoxia-inducible factor (HIF)-1 has been identified as an important transcription factor that mediates the cellular response to hypoxia, promoting both cellular survival and apoptosis under different conditions. Increased tumor cell expression of this transcription factor promotes tumor growth In vivo and is associated with a worse prognosis in patients with non-small-cell lung cancer (NSCLC) undergoing tumor resection. The epidermal growth factor receptor (EGFR) promotes tumor cell proliferation and anglogenesis and inhibits apoptosis. Epidermal growth factor receptor expression increases in a stepwise manner during tumorigenesis and is overexpressed in > 50% of NSCLC tumors. This review discusses the reciprocal relationship between tumor cell hypoxia and EGFR. Recent studies suggest that hypoxia induces expression of EGFR and its ligands. In return, EGFR might enhance the cellular response to hypoxia by increasing expression of HIF-1α, and so act as a survival factor for hypoxic cancer cells. Immunohistochemical studies on a series of resected NSCLC tumors add weight to this contention by demonstrating a close association between expression of EGFR, HIF-1α, and:1 of HIF-1's target proteins, carbonic anhydrase IX. In this article we discuss emerging treatment strategies for NSCLC that target HIF-1, HIF-1 transcriptional targets, and EGFR.

A bioengineered 3D ovarian cancer model for the assessment of peptidase-mediated enhancement of spheroid growth and intraperitoneal spread

Cancer-associated proteases promote peritoneal dissemination and chemoresistance in malignant progression. In this study, kallikrein-related peptidases 4, 5, 6, and 7 (KLK4-7)-cotransfected OV-MZ-6 ovarian cancer cells were embedded in a bioengineered three-dimensional (3D) microenvironment that contains RGD motifs for integrin engagement to analyze their spheroid growth and survival after chemotreatment. KLK4-7-cotransfected cells formed larger spheroids and proliferated more than controls in 3D, particularly within RGD-functionalized matrices, which was reduced upon integrin inhibition. In contrast, KLK4-7-expressing cell monolayers proliferated less than controls, emphasizing the relevance of the 3D microenvironment and integrin engagement. In a spheroid-based animal model, KLK4-7-overexpression induced tumor growth after 4 weeks and intraperitoneal spread after 8 weeks. Upon paclitaxel administration, KLK4-7-expressing tumors declined in size by 91% (controls: 87%) and showed 90% less metastatic outgrowth (controls: 33%, P<0.001). KLK4-7-expressing spheroids showed 53% survival upon paclitaxel treatment (controls: 51%), accompanied by enhanced chemoresistance-related factors, and their survival was further reduced by combination treatment of paclitaxel with KLK4/5/7 (22%, P=0.007) or MAPK (6%, P=0.006) inhibition. The concomitant presence of KLK4-7 in ovarian cancer cells together with integrin activation drives spheroid formation and proliferation. Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient for late-stage disease than chemotherapeutics alone as these inhibitory regimens reduced cancer spheroid growth to a greater extent than paclitaxel alone.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Bcl-2 genes and growth factors in the pathology of ischaemic acute renal failure

For the past decade, an attempt has been made by many research groups to define the roles of the growing number of Bcl-2 gene family proteins in the apoptotic process. The Bcl-2 family consists of pro-apoptotic (or cell death) and anti-apoptotic (or cell survival) genes and it is the balance in expression between these gene lineages that may determine the death or survival of a cell. The majority of studies have analysed the role/s of the Bcl-2 genes in cancer development. Equally important is their role in normal tissue development, homeostasis and non-cancer disease states. Bcl-2 is crucial for normal development in the kidney, with a deficiency in Bcl-2 producing such malformation that renal failure and death result. As a corollary, its role in renal disease states in the adult has been sought. Ischaemia is one of the most common causes of both acute and chronic renal failure. The section of the kidney that is most susceptible to ischaemic damage is the outer zone of the outer medulla. Within this zone the proximal tubules are most sensitive and often die by necrosis or desquamate. In the distal nephron, apoptosis is the more common form of cell death. Recent results from our laboratory have indicated that ischaemia-induced acute renal failure is associated with up-regulation of two anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) in the damaged distal tubule and occasional up-regulation of Bax in the proximal tubule. The distal tubule is a known reservoir for several growth factors important to renal growth and repair, such as insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). One of the likely possibilities for the anti-cell death action of the Bcl-2 genes is that the protected distal cells may be able to produce growth factors that have a further reparative or protective role via an autocrine mechanism in the distal segment and a paracrine mechanism in the proximal cells. Both EGF and IGF-1 are also up-regulated in the surviving distal tubules and are detected in the surviving proximal tubules, where these growth factors are not usually synthesized. As a result, we have been using in vitro methods to test: (i) the relative sensitivities of renal distal and proximal epithelial cell populations to injury caused by mechanisms known to act in ischaemia-reperfusion; (ii) whether a Bcl-2 anti-apoptotic mechanism acts in these cells; and (iii) whether an autocrine and/or paracrine growth factor mechanism is initiated. The following review discusses the background to these studies as well as some of our preliminary results.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

2. Contact lens care and ocular surface homeostasis

Moderator Opening The early focus of contact lens wear and ocular health was on oxygen delivery. However, as we learn more about how the eye works, and investigate how the contact lens interacts with the cornea, the role of the tear film has risen in prominence. A healthy tear film is critical for normal ocular homeostasis, and abnormalities of the tear film are the primary cause of dry eye. In order to improve patient eye health and comfort during lens wear, we need to further elucidate the relationship among contact lenses, contact lens solutions, the tear film, and the corneal epithelium, and find ways to maintain homeostasis of the ocular surface. In this section, we review the latest data and opinions on this complex relationship between contact lenses and lens care solutions

Ocular surface health with contact lens wear

Eye care practitioners (ECPs) would tend to agree that wearing contact lenses increases the risk for infection, but millions of patients are still fitted with lenses every year because ECPs feel that the risk is manageable and that their patients' eye health can be protected. The Fusarium and Acanthamoeba keratitis outbreaks of years past were a wake-up call to manufacturers, ECPs, and regulatory agencies that risk cannot be managed without diligence, and that the complex relationship between contact lens materials, contact lens solutions, and compliance needs to be better understood in order to optimize the efficacy of contact lens care and improve care guidelines.

The TFOS International Workshop on Contact Lens Discomfort: report of the contact lens interactions with the ocular surface and adnexa subcommittee

The report of this subcommittee concerns the impact of contact lenses (CLs) on the ocular surface, with a particular emphasis on CL discomfort (CLD). We define the ocular surface, its regional anatomy, and the physiological responses of each region to CL wear.

In vivo confocal microscopy of the ocular surface : from bench to bedside

In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Diurnal variations in ocular aberrations of human eyes

Purpose: To investigate the diurnal variations in ocular wavefront aberrations over two consecutive days in young adult subjects. Materials and methods: Measurements of both lower-order (sphero-cylindrical refractive powers) and higher-order (3rd and 4th order aberration terms) ocular aberrations were collected for 30 young adult subjects at ten different times over two consecutive days using a Hartmann-Shack aberrometer. Fifteen subjects were myopic and 15 were emmetropic. Five sets of measurements were collected each day at approximately 3 hourly intervals, with the first measurement taken at ~9 am and the final measurement at ~9 pm. Results: Spherical equivalent refraction (p = 0.029) and spherical aberration (p = 0.043) were both found to undergo significant diurnal variation over the two measurement days. The spherical equivalent was typically found to be at a maximum (i.e. most hyperopic) at the morning measurement, with a small myopic shift of 0.37 ± 0.15 D observed over the course of the day. The mean spherical aberration of all subjects (0.038 ± 0.048 μm) was found to be positive during the day and gradually became more negative into the evening, with a mean amplitude of change of 0.036 ± 0.02 μm. None of the other considered sphero-cylindrical refractive power components or higher-order aberrations exhibited significant diurnal variation over the two days of the experiment (p>0.05). Except for the lower-order astigmatism at 90/180 deg (p = 0.040), there were no significant differences between myopes and emmetropes in the magnitude and timing of the observed diurnal variations (p>0.05). Conclusions: Significant diurnal variations in spherical equivalent and spherical aberration were consistently observed over two consecutive days of measurement. Research and clinical applications requiring precise refractive error and wavefront measurements should take these diurnal changes into account when interpreting wavefront data.

High-growth firms : characteristics, job contribution and method observations

Largely as a result of mass unemployment problems in many European countries, the dynamics of job creation has in recent years attracted increased interest on the part of academics as well as policy-makers. In connection to this, a large number of studies carried out in various countries have concluded that SMEs play a very large and/or growing role as job creators (Birch, 1979; Baldwin and Picot, 1995; Davidsson, 1995a; Davidsson, Lindmark and Olofsson, 1993; 1994; 1995; 1997a; 1997b; Fumagelli and Mussati, 1993; Kirchhoff and Phillips, 1988; Spilling, 1995; for further reference to studies carried out in a large number of countries see also Aiginger and Tichy, 1991; ENSR, 1994; Loveman and Sengenberger, 1991; OECD, 1987; Storey and Johnson, 1987). While most researchers agree on the importance of SMEs, there is some controversy as regards whether this is mainly a result of many small start-ups and incremental expansions, or if a small minority of high growth SMEs contribute the lion’s share of new employment. This is known as the ‘mice vs. gazelles’ or ‘flyers vs. trundlers’ debate. Storey strongly advocates the position that the small group of high growth SMEs are the ‘real’ job creators (Storey, 1994; Storey & Johnson, 1987), whereas, e.g., the Davidsson et al research in Sweden (cf. above) gives more support for the ‘mice’ hypothesis.

Identification of a long non-coding RNA gene, GHSROS (growth hormone secretagogue receptor opposite strand), which stimulates cell migration in non-small cell lung cancer cell lines

The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.