J. Guy Lyons

Snail Up-regulates Proinflammatory Mediators and Inhibits Differentiation in Oral Keratinocytes

The transcriptional repressor Snail2 is overexpressed in head and neck squamous cell carcinomas (HNSCC) relative to nonmalignant head and neck mucosal epithelium, and in locally recurrent relative to nonrecurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of proinflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappaB transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin (IL)-6, IL8, or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Reexpression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.

Inflammatory Doses of UV May Not Be Necessary for Skin Carcinogenesis

The UV wavelengths in sunlight are the main cause of skin cancer in humans. Sunlight causes gene mutations, immunosuppression and, at higher doses, inflammation. While it is clear that immunosuppression and gene mutations are essential biologic events via which UV causes skin cancer, the requirement for UV‐induced inflammation is less certain. Both the UVB (290–320 nm) and UVA (320–400 nm) wavebands within sunlight can cause skin cancer, gene mutations and immunosuppression. However, UVB, but not UVA, at realistic doses can cause inflammation, and UVB induces skin cancer, immunosuppression and gene mutations at doses much lower than those required to cause inflammation. Inflammation enhances skin carcinogenesis, but may not be UV induced, and inflammatory mediators at doses too low to cause inflammation may be required. UV‐induced mutations can cause epidermal cells to make proinflammatory factors or to induce them in the surrounding stroma, creating an oxidizing environment in which additional oncogenic mutations are likely to take place, even in the absence of UV. Our hypothesis is therefore that subinflammatory doses of both UVA and UVB cause benign skin tumors. One of the effects of sunlight‐induced mutations may be the production of inflammatory mediators that enhance carcinogenesis.

Mesangial cell-derived factors alter monocyte activation and function through inflammatory pathways: possible pathogenic role in diabetic nephropathy

Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-alpha, IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-alpha, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P < 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P < 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P < 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-alpha, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P < 0.05), but only TNF-alpha and IL-6 increased MMP-9 expression (50- and 60-fold, respectively; P < 0.05). Our results show that mesangial cell-secreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy.

Clonal diversity in carcinomas: its implications for tumour progression and the contribution made to it by epithelial- mesenchymal transitions

The progression of tumours to malignancy is commonly considered to arise through lineal evolution, a process in which mutations conferring pro-oncogenic cellular phenotypes are acquired by a succession of ever-more dominant clones. However, this model is at odds with the persistent polyclonality observed in many cancers. We propose that an alternative mechanism for tumour progression, called interclonal cooperativity, is likely to play a role at stages of tumour progression when mutations cause microenvironmental changes, such as occur with epithelial-mesenchymal transitions (EMTs). Interclonal cooperativity occurs when cancer cell–cancer cell interactions produce an emergent malignant phenotype from individually non-malignant clones. In interclonal cooperativity, the oncogenic mutations occur in different clones within the tumour that complement each other and cooperate in order to drive progression. This reconciles the accepted genetic and evolutionary basis of cancers with the observed polyclonality in tumours. Here, we provide a conceptual basis for examining the importance of cancer cell–cancer cell interactions to the behaviour of tumours and propose specific mechanisms by which clonal diversity in tumours, including that provided by EMTs, can drive the progression of tumours to malignancy.

A spectrophotometric collagenase assay

A quantitative collagenase assay using Coomassie blue staining and microtiter spectrophotometry is described. Collagen is gelled and dried onto the bottom of microwells as substrate, washed, incubated with samples, washed again, and then stained. Absorbance at 590 nm increases linearly with increasing amounts of collagen in the range 5-40 micrograms. Bacterial and mammalian collagenases can be detected within 2 h, and 10 ng of bacterial collagenase may be detected in 16 h. For simple screening applications, activity may be detected by eye. The assay is safe, simple, fast, economical, and sensitive.

Mast Cells Produce Novel Shorter Forms of Perlecan That Contain Functional Endorepellin: A ROLE IN ANGIOGENESIS AND WOUND HEALING*

Background: Mast cells modulate events in wound healing. Results: Shorter forms of perlecan are produced by mast cells via proteolytic processing and alternative splicing, which contain domain V and functional endorepellin. Conclusion: The production of these shorter forms modulates endothelial cell adhesion, proliferation, and migration. Significance: Mast cells produce specific forms of perlecan that affect endothelial cell behavior. Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α2β1 integrin and stimulate the migration of cells in “scratch assays,” both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.

Radiosensitization of Oropharyngeal Squamous Cell Carcinoma Cells By Human Papillomavirus 16 Oncoprotein E6*I

PURPOSE Patients with oropharyngeal squamous cell carcinoma (OSCC) whose disease is associated with high-risk human papillomavirus (HPV) infection have a significantly better outcome than those with HPV-negative disease, but the reasons for the better outcome are not known. We postulated that they might relate to an ability of HPV proteins to confer a better response to radiotherapy, a commonly used treatment for OSCC. METHODS AND MATERIALS We stably expressed the specific splicing-derived isoforms, E6∗I and E6∗II, or the entire E6 open reading frame (E6total), which gives rise to both full length and E6∗I isoforms, in OSCC cell lines. Radiation resistance was measured in clonogenicity assays, p53 activity was measured using transfected reporter genes, and flow cytometry was used to analyze cell cycle and apoptosis. RESULTS E6∗I and E6total sensitized the OSCC cells to irradiation, E6∗I giving the greatest degree of radiosensitization (approximately eightfold lower surviving cell fraction at 10 Gy), whereas E6∗II had no effect. In contrast to radiosensitivity, E6∗I was a weaker inhibitor than E6total of tumor suppressor p53 transactivator activity in the same cells. Flow cytometric analyses showed that irradiated E6∗I expressing cells had a much higher G2M:G1 ratio than control cells, indicating that, after G2, cells were diverted from the cell cycle to programmed cell death. CONCLUSION This study supports a role for E6∗I in the enhanced responsiveness of HPV-positive oropharyngeal carcinomas to p53-independent radiation-induced death.

SWI/SNF: A chromatin-remodelling complex with a role in carcinogenesis

SWI/SNF is a chromatin-remodelling complex that makes DNA that has been compacted into nucleosomes accessible to transcription factors and repair enzymes. It does this by displacing DNA from the core histone surface. SWI/SNF consists of at least nine subunits, including one of two alternative ATPase subunits, BRM or BRG-1, that provide the energy for remodelling. As it regulates access to DNA it controls many aspects of normal cellular function. Limited studies have recently linked loss of function of SWI/SNF subunits to cancer development, suggesting that it may be a tumor suppressor complex. As epigenetic repression regulates SWI/SNF component expression at least in some cases, restoration of function is therapeutically promising for cancer treatment. Considerably more research is required into deregulation of SWI/SNF in cancer and determination of how this affects tumor development. This is an exciting but poorly understood molecule that may have a role in carcinogenesis.

Hotspot Mutation of Brahma in Non-Melanoma Skin Cancer

Mammalian SWItch/sucrose non fermentable (SWI/SNF) remodeling of chromatin modulates transcription and DNA repair. The Brahma (BRM) catalytic subunit of the SWI/SNF complex is one of two mutually exclusive subunits that provide energy for remodeling. BRM has been identified as an important cancer susceptibility locus; however, to date no mutations have been identified in the BRM gene. We performed genetic analysis of BRM in human non-melanoma skin cancers, precancerous lesions, and normal skin revealing a common nonsynonymous point mutation present in one of ten squamous cell and two of six basal cell carcinoma of the skin. This hotspot was not present in germ-line DNA from the same patients, nor in epithelial precancerous lesions. The observed G:C to T:A transversion is typical of mutations occurring following oxidative damage, such as that caused by UVA radiation. This previously unreported hotspot mutation occurs in a highly conserved region of the BRM gene.

Activation of Macrophage Promatrix Metalloproteinase-9 by Lipopolysaccharide-Associated Proteinases

LPS induces an up-regulation of promatrix metalloproteinase-9 (proMMP9) gene expression in cells of the monocyte/macrophage lineage. We demonstrate here that LPS preparations are also able to activate proMMP9 made by human macrophages or THP-1 cells via LPS-associated proteinases, which cleave the N-terminal propeptide at a site or sites close to the one cleaved upon activation with organomercurial compounds. LPS-associated proteinases are serine proteinases that are able to cleave denatured collagens (gelatin) and the mammalian serine proteinase inhibitor, α1-proteinase inhibitor, thereby pushing the balance of extracellular matrix turnover even further toward degradation. A low molecular mass, low affinity inhibitor of MMP9, possibly derived from the propeptide, is generated during proMMP9 activation. However, inhibition of the LPS-associated proteinases had no effect on proMMP9 synthesis, indicating that their proteolytic activity was not required for signaling the up-regulation of the proMMP9 gene.