Perlecan Synthesis Research Articles

Lead suppresses perlecan expression via EGFR-ERK1/2-COX-2-PGI2 pathway in cultured bovine vascular endothelial cells.

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway-the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead-as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.

Cell density-dependent modulation of perlecan synthesis by dichloro(2,9-dimethyl-1,10-phenanthroline)zinc(II) in vascular endothelial cells.

Proteoglycans that are synthesized by vascular endothelial cells contribute to the proliferation, migration, and blood coagulation-fibrinolytic system in vascular endothelial cells. Clarification of the molecular mechanisms for proteoglycan synthesis allows understanding of the regulation of endothelial functions. The research strategy of bioorganometallics analyzes biological systems using organic-inorganic hybrid molecules as tools. The present study found dichloro(2,9-dimethyl-1,10-phenanthroline)zinc(II) and its ligand-modulated perlecan expression in vascular endothelial cells, which depends on the cell density.

Oxygen–glucose deprivation (OGD) and interleukin-1 (IL-1) differentially modulate cathepsin B/L mediated generation of neuroprotective perlecan LG3 by neurons

Brain extracellular matrix (ECM) is highly degraded after cerebral ischemia. The perlecan c-terminal fragment LG3 is generated at increased levels by proteolytic processing as long as 3days after ischemia. It has previously been shown that oxygen–glucose deprivation (OGD), reperfusion and interleukin-1 α (IL-1α) stimulate brain cells to yield increased levels of LG3. This LG3, in turn, is neuroprotective against OGD, and may therefore represent one of the brain's defenses against ischemic injury. Here, we investigate whether, in neurons, this increased LG3 is the result of increased perlecan generation and cellular release, increased protease release (to generate LG3 from previous extracellularly deposited perlecan) or both. We found that pre-synthesized perlecan may be exocytosed by neurons during OGD and de novo synthesis of perlecan is increased during reperfusion, even 24h after OGD. Furthermore, while cathepsin L activity was seen to be marginally important to generate LG3 during normoxic conditions, cathepsin B activity was found to be important to generate increased levels of LG3 following OGD and reperfusion. On the other hand, IL-1α treatment raised levels of cathepsin L in neuronal media, and both cathepsin L and cathepsin B were demonstrated to be important for increasing LG3 levels after IL-1α treatment.

A perlecan-inducing compound significantly inhibits smooth muscle cell function and in-stent intimal hyperplasia: novel insights into the diverse biological effects of perlecan

Perlecan is the major heparan sulfate proteoglycan in the arterial wall. Previous studies have suggested that perlecan is a potent inhibitor of smooth muscle cell (SMC) activity. Therefore, perlecan overexpression may serve as a therapeutic modality to prevent in-stent restenosis (ISR). We have investigated a novel compound (RUS3108), identified in a SMC based screen to induce perlecan synthesis in SMC. The aims of this study were to assess the in vitro effects of RUS3108 and the effects of RUS3108-eluting stents in preventing ISR.Rabbit aortic SMC and bovine aortic endothelial cells (EC) were used in this study. Immunohistochemistry showed that RUS3108-treated SMC over-expressed perlecan indicating the drug effects. Furthermore, RUS3108 induced a SMC differentiated phenotype by SMembryonic staining. RUS3108 (1 microM) inhibited 3H-thymidine incorporation by >50%, which was completely reversed by a perlecan antibody. RUS3108 also inhibited SMC migration (Boyden chamber) and MMP-9 activity. In contrast, RUS3108 (100nM) modestly stimulated EC 3H-thymidine incorporation by 22% (p<0.02). In vivo, a total of 30 stents were deployed in rabbit iliac arteries as follows: 1) bare metal stents (n=10), 2) polymer onlycoated stents (n=10), and 3) polymer-coated stents containing RUS3108 (n=10). Rabbits were sacrificed at four weeks and stented segments were subjected to morphometric analysis. Intimal cross sectional area was significantly lower in the RUS3108-eluting stent group (0.31+ or -0.27 mm(2) versus 1.0 + or - 0.31 and 1.25 + or - 0.51 in the bare metal stents and polymer only coated stents groups, respectively, p<0.0001).RUS3108 is a novel perlecan-inducing compound, which is a potent inhibitor of SMC activity and a modest stimulator of EC proliferation. RUS3108-eluting stents may serve as an excellent modality for the prevention of ISR.

The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells

A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer.

Changes in perlecan during chondrocyte differentiation in the fetal bovine rib growth plate

Perlecan is a heparan sulfate proteoglycan present in the growth plate and essential for endochondral ossification. We evaluated the synthesis and structure of perlecan in the different zones of the growth plate. The growth plates from fetal bovine ribs were isolated and sequentially sliced into 1-mm sections containing the hypertrophic zone, lower proliferative zone, upper proliferative zone, intermediate zone, and resting zone, respectively. The slices were then either incubated in culture medium with 35SO4 to measure total sulfated proteoglycan synthesis and perlecan synthesis, extracted for perlecan core protein analysis by Western blot, or extracted for perlecan isolation and subsequent characterization of glycosaminoglycan size and disaccharide composition. 35SO4 incorporation into perlecan was three-fourfold higher in the proliferating/hypertrophic zone than the resting zone. Western blot showed perlecan content was greatest in the lower and upper proliferating zones and that a perlecan fragment lacking portions of the N- and C-terminal domains containing heparan sulfate was also present in all zones. Purified perlecan from the hypertrophic/lower proliferative zone had larger chondroitin sulfate chains and a different composition of CS and HS disaccharides than the perlecan isolated from the resting zone. These results indicate perlecan deposition is increased and is turned over during proliferation to be replaced by a perlecan with a different sulfation pattern.

Cell Biology of Heavy Metal Toxicity in Vascular Tissue

AbstractFor Abstract see ChemInform Abstract in Full Text.

Reduction of perlecan synthesis and induction of TGF-beta1 in human peritoneal mesothelial cells due to high dialysate glucose concentration: implication in peritoneal dialysis.

Prolonged exposure of the peritoneal mesothelium to high dialysate glucose concentrations reduces anionic sites that are critical to its selective permeability, thereby impairing the peritoneal transport properties in patients on long-term peritoneal dialysis (PD). Perlecan, an anionic heparan sulfate proteoglycan, is pivotal to the selective permeability of basement membranes, and high glucose concentrations modulate its synthesis in mesangial cells. The effect of glucose on perlecan expression in the peritoneal mesothelium has not been established. We investigated perlecan expression in peritoneal biopsies from patients on PD, and the effect of high glucose concentrations on perlecan synthesis in cultured human peritoneal mesothelial cells (HPMC). Peritoneal biopsies from PD patients showed reduced perlecan expression compared with controls. Exposure of HPMC to high glucose concentrations resulted in a dose-dependent reduction in the synthesis of perlecan polypeptide and its deposition into the extracellular matrix. These effects were mediated in part through the induction of TGF-beta1. Characterization studies showed that perlecan synthesized by HPMC contained solely heparan sulfate glycosaminoglycan (HS GAG) chains, and [(35)S]-incorporation studies demonstrated progressive reduction of their de novo synthesis with increasing glucose concentrations (68142 +/- 3658, 48147 +/- 2517, 31468 +/- 5781, and 25575 +/- 3621 cpm/ micro g cellular protein for 5 mM, 30 mM, 75 mM, and 120 mM D-glucose, respectively; P < 0.001 for 5 mM versus 30 mM D-glucose, and P < 0.0001 for 5 mM versus 75 mM or 120 mM D-glucose). Both the length and the charge density of the HS GAG chains remained unchanged. Reduction of peritoneal perlecan expression in long-term PD was attributed to high dialysate glucose concentrations, which induced TGF-beta1 and reduced perlecan synthesis in HPMC. Since perlecan can sequester growth factors, thereby modulating cell migration and differentiation perturbation of peritoneal perlecan expression contributes to the structural and functional changes of the peritoneum in long-term PD.

Cell biology of heavy metal toxicity in vascular tissue

Cadmium and lead are heavy metals that have been shown to induce vascular disorders such as atherosclerosis in experimental animals. However, little is known about the mechanisms by which cadmium and lead induce vascular toxicity. The toxicity was investigated using a culture system of vascular endothelial and smooth muscle cells. Cadmium destroys the monolayer of endothelial cells and the cytotoxicity is protected by zinc and copper without metallothionein induction. On the other hand, lead does not exhibit cytotoxicity but inhibits the repair of endothelial monolayers after wounding by a lower response to endogenous basic fibroblast growth factor mediated by suppression of the synthesis of perlecan, a large heparan sulfate proteoglycan. In addition, cadmium and lead reduce endothelial fibrinolytic activity by induction of plasminogen activator inhibitor type 1 synthesis and by inhibition of tissue-type plasminogen activator, respectively. In vascular smooth muscle cells, cadmium and lead can promote their proliferation and influence proteoglycan synthesis and fibrinolysis in different manners. These results indicate that cadmium and lead have specific toxicities in the proliferation, fibrinolysis, and extracellular matrix formation of vascular endothelial and smooth muscle cells.

Regulation of Glomerular Endothelial Cell Proteoglycans by Glucose

The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.

Cell Biological Study on Abnormal Proteoglycan Synthesis in Vascular Cells Exposed to Heavy Metals

Lead and cadmium are toxic heavy metals that have been shown to be possible risk factors of atherosclerosis in epidemiological and experimental studies. Since excess extracellular matrix, including proteoglycans, accumulates changing the composition and the structure in the atherosclerotic vascular wall, the effects of lead and cadmium on the proteoglycan synthesis in vascular cells have been studied using a cell culture system. The following results were obtained: Lead does not destroy endothelial cell layers but markedly inhibits the repair of the injured cell layers, which results from a lower response to endogenous basic fibroblast growth factor caused by inhibition of the synthesis of perlecan, a large heparan sulfate proteoglycan, in vascular endothelial cells. Lead selectively inhibits the synthesis of versican, a large chondroitin sulfate proteoglycan, in vascular smooth muscle cells only at a high cell density. Cadmium induces the synthesis of biglycan and decorin, small dermatan sulfate proteoglycans, in vascular smooth muscle cells only at a low cell density, while inhibiting the synthesis of other proteoglycan molecules. It is therefore suggested that lead and cadmium may influence the developmental process of atherosclerosis through disrupting the regulation of proteoglycan synthesis in vascular cells.

Disaccharide Composition of Glycosaminoglycan Chains in Growing Vascular Endothelial Cells in Culture after Exposure to Lead

Heparan sulfate (HS) chains bind and activate fibroblast growth factor-2 (FGF-2) depending on their microstructure. The glycosaminoglycan chains present particularly as a large HS proteoglycan perlecan in vascular endothelial cells. A heavy metal lead induces a lower response to FGF-2 by inhibition of perlecan synthesis and inhibits the proliferation of the cells. The present study was undertaken to address the question whether lead influences the formation of HS chains in vascular endothelial cells. The data indicate that lead decreases the amount of disaccharide units including hexuronic acid-N-acetylglucosamine, hexuronic acid-N-sulfated glucosamine and 2-O-sulfated hexuronic acid-N-sulfated glucosamine in HS chains accumulated in the cell layer and the conditioned medium. However, no percentage of any disaccharide unit was affected by the metal. The percentage of disaccharide units in chondroitin/dermatan sulfate was also unaffected by lead. The present data support the hypothesis that lead-induced lower response of vascular endothelial cells to endogenous FGF-2 mainly results from a decrease in perlecan molecules of extracellular matrix as a result of selective inhibition of perlecan core protein synthesis rather than a change in the HS structure.

Syndecan-3 and perlecan are differentially expressed by progenitors and mature oligodendrocytes and accumulate in the extracellular matrix.

Oligodendrocyte progenitors originate in the subventricular zone, proliferate, migrate to their final destinations, differentiate, and interact with axons to produce multilamellar myelin sheaths. These processes are regulated by a variety of environmental signals, including growth factors, the extracellular matrix, and adhesion molecules. Heparan sulfate proteoglycans are premier candidates as participants in this regulation by virtue of their structural diversity and their capacity to function as coreceptors for both growth factors and extracellular matrix molecules. Consistently with this, we have previously shown that oligodendrocyte progenitors are unable to proliferate in response to fibroblast growth factor-2 (FGF-2) in the absence of sulfated heparan sulfate proteoglycan. Here we show that members of three families of heparan sulfate proteoglycans, syndecan, perlecan, and glypican, are developmentally and posttranscriptionally regulated during oligodendrocyte-lineage progression: Syndecan-3 is synthesized by oligodendrocyte progenitors (but not terminally differentiated oligodendrocytes) and is up-regulated by FGF-2; perlecan synthesis increases as oligodendrocytes undergo terminal differentiation; glypican-1 is expressed by both progenitors and differentiated oligodendrocytes. Astrocytes express glypican-1 and perlecan but not syndecan-3. All three of these heparan sulfate proteoglycans are shed from the cell surface and bind to specific substrates. The developmentally regulated expression of these heparan sulfate proteoglycans is indicative of their participation in events involving growth factor receptors and the extracellular matrix that may regulate oligodendrocyte progenitor proliferation, migration, and adhesion phenomena.

Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity.

Perlecan, a proteoglycan of basement membrane and extracellular matrices, has important roles in both normal biological and pathological processes. As a result of its ability to store and protect growth factors, perlecan may have crucial roles in tumour-cell growth and invasion. Since the biological functions of different types of glycosaminoglycan vary with cellular origin and structural modifications, we analysed the expression and biological functions of perlecan produced by a normal epidermal cell line (JB6) and its transformed counterpart (RT101). Expression of perlecan in tumorigenic cells was significantly increased in both mRNA and protein levels. JB6 perlecan was exclusively substituted with heparan sulphate, whereas that of RT101 contained some additional chondroitin sulphate. Detailed structural analysis of the heparan sulphate (HS) chains from perlecan of both cell types revealed that their overall sulphation and chain length were similar (approximately 60 kDa), but the HS chains of tumour-cell-derived perlecan were less sulphated. This resulted from reduced 2-O- and 6-O-sulphation, but not N-sulphation, and an increase in the proportion of unsulphated disaccharides. Despite this, the heparan sulphate of RT101- and JB6-derived perlecan bound fibroblast growth factor-1, -2, -4 and -7 and heparin-binding epidermal growth factor with similar affinity. Therefore abundant tumour-derived perlecan may support the angiogenic responses seen in vivo and be a key player in tumorigenesis.

Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity

Perlecan, a proteoglycan of basement membrane and extracellular matrices, has important roles in both normal biological and pathological processes. As a result of its ability to store and protect growth factors, perlecan may have crucial roles in tumour-cell growth and invasion. Since the biological functions of different types of glycosaminoglycan vary with cellular origin and structural modifications, we analysed the expression and biological functions of perlecan produced by a normal epidermal cell line (JB6) and its transformed counterpart (RT101). Expression of perlecan in tumorigenic cells was significantly increased in both mRNA and protein levels. JB6 perlecan was exclusively substituted with heparan sulphate, whereas that of RT101 contained some additional chondroitin sulphate. Detailed structural analysis of the heparan sulphate (HS) chains from perlecan of both cell types revealed that their overall sulphation and chain length were similar (≈ 60kDa), but the HS chains of tumour-cell-derived perlecan were less sulphated. This resulted from reduced 2-O- and 6-O-sulphation, but not N-sulphation, and an increase in the proportion of unsulphated disaccharides. Despite this, the heparan sulphate of RT101- and JB6-derived perlecan bound fibroblast growth factor-1, -2, -4 and -7 and heparin-binding epidermal growth factor with similar affinity. Therefore abundant tumour-derived perlecan may support the angiogenic responses seen in vivo and be a key player in tumorigenesis.

Glycosaminoglycans: use in treatment of diabetic nephropathy.

Glycosaminoglycans: use in treatment of diabetic nephropathy.

Inhibition of the Repair of Injured Endothelial Cell Monolayers by Lead and Its Possible Mechanisms

Lead is a heavy metal which has been experimentally and epidemiologically shown to induce vascular lesions such as atherosclerosis, however, little is known about the mechanism. Since damage of endothelial cell layers is a key event in the initiation of atherosclerosis, the effect of lead on the maintenance of cell layers has been studied using a cell culture system in our laboratory. The following results were obtained : (1) Endothelial cell monolayers are not destroyed but the repair after injury is markedly inhibited by lead through inhibition of the proliferation. (2) Lead suppresses the proliferative response of endothelial cells to basic fibroblast growth factor (bFGF). (3) Lead reduces the amount of endogenous bFGF bound to heparan sulfate proteoglycans through inhibition of the synthesis of perlecan, a large heparan sulfate proteoglycan. Since heparan sulfate chains of perlecan promote the binding of bFGF to its receptor, our results indicate that inhibition of the repair of injured endothelial monolayers by lead is due to a lower proliferative response of the cells to endogenous bFGF, caused by a suppression of perlecan synthesis.

Regulation of the heparan sulfate proteoglycan, perlecan, by injury and interleukin-1alpha.

Perlecan is a specific proteoglycan that binds to amyloid precursor protein and beta-amyloid peptide, is present within amyloid deposits, and has been implicated in plaque formation. Because plaque formation is associated with local inflammation, we hypothesized that the mechanisms involved in brain inflammatory responses could influence perlecan biosynthesis. To test this hypothesis, we first studied perlecan regulation in mice after inflammation induced by a brain stab wound. Perlecan mRNA and immunoreactivity were both increased 3 days after injury. Interleukin-1alpha (IL-1alpha) is a cytokine induced after injury and plays an important role in inflammation. As such, IL-1alpha may be one of the factors participating in regulating perlecan synthesis. We thus studied perlecan regulation by IL-1alpha, in vivo. Regulation of perlecan mRNA by this cytokine was area-specific, showing up-regulation in hippocampus, whereas in striatum, perlecan mRNA was unchanged. To support this differential regulation of perlecan mRNA by IL-1alpha, basic fibroblast growth factor (bFGF), a growth factor also present in plaques, was studied in parallel. bFGF mRNA did not show any regional difference, being up-regulated in both hippocampus and striatum in vivo. In vitro, both astrocyte and microglia were immunoreactive for perlecan. Moreover, perlecan mRNA was increased in hippocampal glial cultures after IL-1alpha but not in striatal glia. These results show an increase in perlecan biosynthesis after injury and suggest a specific regulation of perlecan mRNA by IL-1alpha, which depends on brain area. Such regulation may have important implications in the understanding of regional brain variations in amyloid plaque formation.

Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture

We characterized proteoglycans synthesized by growing cultured bovine aortic endothelial cells after exposure to lead. Lead significantly decreased the incorporation of both [ 3H]glucosamine and [ 35S]sulfate into glycosaminoglycans accumulated in the cell layer and the conditioned medium of the cells in a dose-dependent manner. Proteoglycans metabolically labeled with [ 35S]sulfate in the presence of lead were separated into heparan sulfate proteoglycans (HSPGs) and more highly charged chondroitin/dermatan sulfate proteoglycans by DEAE-Sephacel ion-exchange chromatography. It was found that lead markedly inhibited the synthesis of HSPGs. Sepharose CL-4B molecular sieve gel filtration showed that the marked decrease by lead occurred in the high molecular weight subclass of HSPGs. However, the length of heparan sulfate chains (approximately 50 kDa) was unchanged when analyzed by Sepharose CL-6B chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of core proteins showed that lead reduced the accumulation of a high molecular weight (approximately 400 kDa) HSPG core protein in the cell layer and the conditioned medium; the core protein was identified as a perlecan core by Western blot analysis. It is suggested that lead inhibits the synthesis of the perlecan core protein in growing endothelial cells without a change of heparan sulfate chain length. The present data support the hypothesis that inhibition of endothelial cell proliferation by lead may result from a lower response of the cells to endogenous basic fibroblast growth factor whose binding to the receptor is strongly promoted by heparan sulfate chains of perlecan.

Suppression of invasive behavior of melanoma cells by stable expression of anti-sense perlecan cDNA

Heparan sulfate proteoglycans are one of the major components of extracellular matrix and are secreted at different levels by several normal and tumoral cells. Perlecan, the basement membrane proteoglycan, has structural domains involved in cell/matrix interactions and growth factor storage. Metastatic melanoma cells show an increase in perlecan expression as compared to low metastatic ones. We examined whether reduction of perlecan expression could down-modulate the malignant phenotype in melanoma clones. We transfected B16-F10 murine malignant melanoma cells with a perlecan antisense cDNA construct and tested the in vitro behavior of the selected clones. The expression of antisense mRNA corresponded to a reduction of perlecan synthesis. The clones with reduced perlecan synthesis showed a down-regulation of proliferation and invasion. These results further indicate the importance of perlecan as a regulator of growth factor activity affecting the biological properties of metastatic cells, and suggest the potential use of antisense perlecan DNA in anti-melanoma gene therapy approaches.