Michael Edward

The relationship between connexins, gap junctions, tissue architecture and tumour invasion, as studied in a novel in vitro model of HPV-16-associated cervical cancer progression.

Disruption of gap junctional intercellular communication (GJIC) and/or connexins (gap junction proteins) is frequently reported in malignant cell lines and tumours. Certain human papillomaviruses (HPV) associated with the development of cancers, especially of the cervix, have previously been reported to downregulate GJIC in vitro. There is also evidence for reduced gap junctions in cervical dysplasia. However, many squamous hyperproliferative conditions, including HPV-induced warts, often show extensive upregulation of certain connexins. The association between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in cervical tumour progression, remains unclear. Therefore, using a variety of nonmalignant and malignant cell lines and an organotypic raft-culture system, we investigated the relationship between HPV, gap junctions and tumour progression. Established cervical tumour cell lines carrying HPV were unable to communicate via gap junctions (when assayed by dye-transfer techniques). This correlated with lack of connexin protein expression, while transfection with connexins 26 or 43 led to functional gap junction membrane plaques. On the other hand, immortal but nonmalignant cell lines that contained episomal or integrated HPV-16, but required feeder-layer and growth-factor support, were consistently well coupled, and expressed multiple connexins at membrane junctions. In vitro selection of feeder-layer and growth-factor-independent variants eventually lead to loss of GJIC, which correlated with loss of membrane and increased cytoplasmic connexin 43 localization. However, this was preceded by loss of differentiation and stromal invasion, as assayed on the organotypic raft-culture model. Using this model, a comparison between noncoupled, well-coupled and connexin-transfected cell lines revealed no firm correlation between GJIC and dysplasia, but GJIC appeared to favour increased stratification. These findings demonstrate that loss of GJIC is frequent, but appears to occur more as a consequence of, rather than being the cause of, epithelial dysplasia, and may be influenced by, but is not directly attributable to, HPV.

Different susceptibilities of melanoma cells to retinoic acid-induced changes in melanotic expression

The effect of retinoic acid on murine B16 melanoma cell growth, tyrosinase activity and melanin synthesis was investigated. Retinoic acid inhibited the growth of B16F1, B16F10 and B16BL6 melanoma cells, but enhanced melanin synthesis only in the B16F1 cells. The B16F10 and B16BL6 cells exhibited retinoic acid-induced suppression of tyrosinase activity and melanin synthesis, which was most apparent in the B16F10 cell variant. For comparison, Cloudman S91 melanoma cells proved to be particularly sensitive to retinoic acid-induced growth inhibition and stimulation of the expression of their melanotic phenotype. These results suggest considerable heterogeneity in the B16 melanoma with respect to their response to retinoic acid.

Effect of Different Classes of Gadolinium-based Contrast Agents on Control and Nephrogenic Systemic Fibrosis–derived Fibroblast Proliferation

PURPOSE To determine the ability of different types of gadolinium-based contrast agents (GBCAs) to stimulate fibroblast proliferation in monolayer cell culture. MATERIALS AND METHODS The National Health Service West Glasgow Ethics Committee granted approval for this study. Fibroblasts established from healthy volunteers (control subjects) and from lesional skin of patients with nephrogenic systemic fibrosis were exposed to a range of concentrations of ionic and nonionic linear and macrocyclic contrast agents over 4 days, and the effect on growth was determined. The lowest concentration of contrast agent that stimulated the maximum effect on fibroblast growth was selected for determination of its effect on fibroblast growth over 8 days. The effect of contrast agents on hyaluronan and collagen synthesis was determined with an enzyme-linked immunosorbent assay. Responses were assessed with analysis of variance (general linear model). RESULTS The linear gadolinium contrast agents (gadodiamide, gadoversetamide, gadopentetate dimeglumine, and gadobenate dimeglumine) produced a maximum stimulation of fibroblast proliferation at a concentration of 0.1 mmol/L, with cell numbers increasing up to 2.3-fold. The macrocyclic contrast agents (gadoteric acid and gadoteridol) produced a maximum stimulation of fibroblast proliferation at a concentration of 5 mmol/L. The reference gadolinium agents (N-methylglucamine gadolinium ethylenediaminetetraacetic acid and gadolinium trichloride) stimulated fibroblast proliferation at a concentration of 0.01 mmol/L and were toxic at a concentration greater than 1 mmol/L. Growth curves supported the dose-response observations. Hyaluronan synthesis was stimulated by gadoversetamide, gadobenate dimeglumine, gadodiamide, and gadopentetate dimeglumine at a concentration of 0.1 mmol/L and by gadolinium trichloride at a concentration of 0.01 mmol/L, whereas collagen synthesis was unaffected. CONCLUSION This study provides evidence that different classes of gadolinium chelates stimulate human fibroblast proliferation.

Keratinocyte regulation of TGF‐β and connective tissue growth factor expression: A role in suppression of scar tissue formation

Allogeneic keratinocytes applied to large full‐thickness wounds promote healing while suppressing scar tissue formation. This effect may be mediated in part by their effect on the levels of transforming growth factor‐βs (TGF‐βs) and connective tissue growth factor (CTGF) in the wound and subsequent modulation of fibroblast activity. We have examined the levels of TGF‐β and CTGF produced by keratinocytes and fibroblasts, and the effect of keratinocyte‐conditioned medium using monolayer and living skin‐equivalent cultures. Keratinocyte monolayers did not release any detectable TGF‐β1, but released moderate levels of TGF‐β2 into culture medium, and stained strongly for TGF‐β1, but only weakly for TGF‐β2. Fibroblasts released large amounts of TGF‐β1, no TGF‐β2, and stained strongly for TGF‐β1. Neither cell type released TGF‐β3, but both stained strongly for TGF‐β3. Keratinocyte‐conditioned medium suppressed the levels of TGF‐βs and CTGF associated with the fibroblasts compared with fibroblasts incubated in Dulbeccos minimal essential medium and fibroblast‐conditioned medium. In living skin equivalents, keratinocytes stained very strongly for TGF‐β1 and CTGF, moderately strongly for TGF‐β3, and only weakly for TGF‐β2. Fibroblasts stained strongly for TGF‐β1 and 3 and CTGF. These observations suggest that keratinocytes may affect the TGF‐β profile in such a way as to suppress the formation of scar tissue.

Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling

In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRα/β inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth.

Retinoic acid-induced inhibition of metastatic melanoma cell lung colonization and adhesion to endothelium and subendothelial extracellular matrix

The effect of pretreatment of metastatic B16 melanoma cells with 10−6M all trans-retinoic acid resulted in a significant inhibition of lung colonization following injection of 105 cells into the tail vein of syngeneic C57BL mice. Adhesion of melanoma cells to vascular endothelial cell monolayers, and subendothelial extracellular matrix was also inhibited by pretreatment with retinoic acid, as was tumour cell aggregation following seeding of pretreated cells on to 0.5% agar. Release of 35SO4 from radiolabelled subendothelial extracellular matrix by melanoma cells was essentially unaltered by retinoic acid pretreatment, as was the release of radiolabel from [3H]proline-labelled matrix, while plasminogen activator activity was enhanced in retinoic-acid-treated cells. These observed changes in adhesive properties may be responsible, at least in part, for the retinoic-acid-induced inhibition of lung colonization.

Melanoma cell-derived factor stimulation of fibroblast glycosaminoglycan synthesis—The role of platelet-derived growth factor

The hyaluronan-rich matrix surrounding many tumours may facilitate tumour growth, invasion and angiogenesis, with the majority of this hyaluronan apparently being synthesised by normal fibroblasts, stimulated to do so by tumour cell-derived factors. Melanoma cell-conditioned medium (CM) stimulates up to a 6-fold increase in fibroblast glycosaminoglycan (GAG) synthesis, with the active factors being present in tumour CM ultrafiltration fractions > 30 kDa and < 1 kDa. These fractions are poorly active individually, but when recombined, the activity is substantially greater than the additive effect. The objective of this study was to identify the factors present in the ultrafiltration fraction > 30 kDa that produce a greater than additive effect with the fraction < 1 kDa in stimulating the incorporation of 3H glucosamine into fibroblast GAGs. A number of factors including basic fibroblast growth factor (bFGF), interleukin (IL)-1 beta, pleiotrophin, platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF) failed to stimulate any significant increase in GAG synthesis, but when added to the < 1 kDa tumour CM fraction, both PDGF and to a lesser extent, bFGF, exhibited potent stimulating activities. Neutralising antibodies to PDGF and bFGF added to the melanoma CM decreased the fibroblast GAG-stimulating activity by 29% and 40%, respectively, in C8161 melanoma CM and by 47% and 45%, respectively, in Hs294T melanoma CM. The activities of PDGF-AA and PDGF-BB isoforms were indistinguishable, suggesting the PDGF-alpha receptor plays a role in the GAG-stimulatory response. Western analysis following treatment with PDGF, bFGF or melanoma CM revealed banding patterns for PDGF and melanoma CM that were similar. Immunoprecipitation of the PDGF-alpha receptor revealed it to be phosphorylated in fibroblasts treated with PDGF and melanoma CM, but not with control fibroblast CM. These studies suggest that PDGF plays an important role in the GAG-stimulating activity of the melanoma CM, but requires the presence of an as yet unidentified novel low molecular weight factor for full activity.

Keratinocytes stimulate fibroblast hyaluronan synthesis through the release of stratifin: A possible role in the suppression of scar tissue formation

Scar tissue formation during healing of extensive skin wounds may be related to delayed reepithelialization, while elevated levels of hyaluronan may suppress scar tissue formation. This study investigates the expression of hyaluronan by human skin fibroblasts in response to keratinocyte‐conditioned medium (KCM), and attempts to identify any active factors within the conditioned medium. Serum‐free KCM was assessed for its ability to stimulate the incorporation of 3H‐glucosamine into fibroblast glycosaminoglycans, and hyaluronan synthesis. Conditioned medium was concentrated with an ultrafiltration membrane with a 30 kDa cutoff. Stratifin was assessed for its ability to stimulate hyaluronan synthesis and its role in KCM. KCM stimulated fibroblast glycosaminoglycan synthesis up to a 3.3‐fold increase and a 6.5‐fold increase in hyaluronan synthesis compared with serum‐free controls. Preliminary characterization of the active factors showed that they are retained by a >30 kDa ultrafiltration membrane, and are protease sensitive and heat resistant. Emmprin and stratifin were shown to stimulate fibroblast hyaluronan synthesis, and the hyaluronan‐stimulating activity of KCM was removed following stratifin depletion and to a lesser extent by emmprin depletion. Keratinocytes release soluble factors, including stratifin, that stimulate fibroblast hyaluronan synthesis, and this stimulation of fibroblast hyaluronan may contribute to the suppression of scar tissue formation.

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

Partial Characterisation of Human Melanoma Cell-Derived Factors that Stimulate Fibroblast Glycosaminoglycan Synthesis.

Glycosaminoglycans (GAGs), and in particular hyaluronan, are known to play a role in tumour cell migration, invasion and metastasis. Conditioned medium from two human metastatic melanoma cell lines (Hs294T and C8161) shows potent fibroblast GAG-synthesis-stimulating activities which are active in fibroblast cultures derived from different anatomical sites. This ability is not specific to melanoma cells and is observed in several carcinoma cell lines. Initial characterisation studies have demonstrated that the GAG-stimulating activities in the medium conditioned with melanoma cells show a degree of heat and trypsin resistance. Fractionation of the conditioned medium with Amicon ultrafiltration membranes of various molecular weight cut-offs, ranging from 1 to 30 kD, resulted in a total loss of activity. Activity could be regained by recombination of the concentrated fraction with the filtrate, suggesting more than one factor to be involved in GAG stimulation, with a degree of interdependence between the individual fractions. The fraction greater than 30 kD and that less than 1 kD appear to contain the majority of the GAG-stimulating activity.