Mark Nesbit

E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.

In human epidermis, functional symbiosis requires homeostatic balance between keratinocytes and melanocytes. Compelling evidence from co-culture studies demonstrated a sophisticated, multileveled regulation of normal melanocytic phenotype orchestrated by undifferentiated, basal-type keratinocytes. Keratinocytes control cell growth and dendricity, as well as expression of melanoma-associated cell surface molecules of normal melanocytes. In contrast, melanoma cells are refractory to the keratinocyte-mediated regulation. The loss of regulatory dominance by keratinocytes occurs in concert with down-regulation of E-cadherin expression in melanoma cells. To investigate the potential role of E-cadherin in melanoma-keratinocyte interaction, we transduced E-cadherin-negative melanoma cells with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional E-cadherin expression in melanoma cells leads to cell adhesion to keratinocytes rendering them susceptible for keratinocyte-mediated control. In a skin reconstruction model, ectopic E-cadherin expression inhibits invasion of melanoma cells into dermis by down-regulating invasion-related adhesion receptors, MelCAM/MUC18 and beta3 integrin subunit, and by induction of apoptosis. Thus, disruption of the E-cadherin-mediated, normal regulatory control from keratinocytes may represent one of the mechanisms accounting for melanocyte transformation.

Human Melanoma Progression in Skin Reconstructs : Biological Significance of bFGF

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

Low-Level Monocyte Chemoattractant Protein-1 Stimulation of Monocytes Leads to Tumor Formation in Nontumorigenic Melanoma Cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-α, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-α production.

Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes

Basic fibroblast growth factor (bFGF or FGF-2) is produced by nearly all melanomas in vitro and in vivo but not by normal melanocytes, which require exogenous bFGF for growth. In this study, we transduced normal human melanocytes to overexpress two forms of bFGF: (bFGF-Long and bFGF-Short) using replication-deficient adenovirus 5 vectors. bFGF-Long induced the 17.8, 22.5, 23.1 and 24.2u2009kDa forms of bFGF, whereas bFGF-Short induced only the 17.8u2009kDa mature form. Growth of cultured melanocytes transduced with either vector was similar to that of nevus and melanoma cells and was independent of exogenous bFGF and of insulin/insulin-like growth factor 1, and cyclic AMP enhancers, requiring only phorbol ester as an exogenous mitogen. Like primary melanoma cells, transduced normal melanocytes grew anchorage independently in soft agar. When injected into the dermis of human skin grafted to mice, bFGF-transduced melanocytes proliferated for at least 20 days, whereas cells from control cultures showed poor survival and no proliferation. These results demonstrate that bFGF upregulation is a critical component in melanoma progression.

Differential response of primary and metastatic melanomas to neutrophils attracted by IL‐8

IL‐8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL‐8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL‐8 with IL‐8 cDNA using a replication‐deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque‐forming units of IL‐8 per cell. However, at high IL‐8 transduction levels (100 ng/ml/105 cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL‐8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL‐8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL‐8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL‐8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.

Matrix Immobilization Enhances the Tissue Repair Activity of Growth Factor Gene Therapy Vectors

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Interleukin-8 overexpression is present in pyoderma gangrenosum ulcers and leads to ulcer formation in human skin xenografts.

Interleukin-8 (IL-8) is a potent chemotactic polypeptide for neutrophils. However, the role of this cytokine during inflammation remains unclear. Skin specimens from patients with pyoderma gangrenosum demonstrated IL-8 overexpression in skin ulcers, which suggests a role for IL-8 in the development of the disease. We therefore constructed a recombinant adenovirus expressing the complementary deoxyribonucleic acid encoding human IL-8 (IL-8/Ad5) that induces a 2000-fold increase in IL-8 expression of infected human fibroblasts in vitro. Human skin engrafted to severe combined immunodeficiency mice and then injected with the recombinant virus demonstrated erythema, an intense perivascular infiltration of neutrophils, and extravasation of erythrocytes after 8 hours. By 12 hours after injection, neutrophils had accumulated beneath the epidermis, which then necrotized, and one or more ulcers that remained for approximately 2 weeks were observed. Clinically and histologically, the ulcers resembled pyoderma gangrenosum. These clinical and experimental findings suggest an etiologic role of IL-8 in the pathogenesis of pyoderma gangrenosum.

Adenoviral-mediated gene transfer in wound healing: acute inflammatory response in human skin in the SCID mouse model

The use of an adenoviral vector as a means of therapeutic protein delivery for the treatment of impaired wound healing is a potentially effective application of current gene transfer techniques. This study was designed to investigate the ability of adenovirus to mediate gene transfer in healing wounds in human skin in vivo. The human skin/severe combined immunodeficient mouse chimera model was used to study both the response of human tissue to adenoviral infection and the nature of the acute inflammatory response. The effects of adenoviral infection and transgene expression on the rate and quality of human wound healing were then investigated. Cell‐ and species‐specific monoclonal antibodies were used to characterize the resident skin cell types participating in wound repair, the inflammatory response, and the proliferative potential of adenovirus‐treated compared to control skin. Our studies show that, following wounding, normal skin architecture is restored in the presence of adenoviral infection equivalent to noninfected controls. Despite an increased acute inflammatory response after adenovirus injection, no difference in the healing capabilities of wounded skin was observed, suggesting that adenovirus‐mediated gene transfer for growth factor‐mediated acceleration of wound healing may be feasible.

Production of a functional monoclonal antibody recognizing human colorectal carcinoma cells from a baculovirus expression system

The light and heavy chain cDNA of a murine monoclonal antibody (MoAb) with specificity for human colorectal carcinoma cells have been expressed separately, together, and as a dual construct in insect cells infected with recombinant baculoviruses. High levels of the MoAb were expressed under the control of the polyhedrin promoter. The antibody maintained its specific binding to human colorectal carcinoma cells and mediated lysis of these cells by human lymphocytes, monocytes, and murine macrophages, as determined in antibody-directed cellular cytotoxicity (ADCC) assays. The recombinant immunoglobulin (Ig), like its ascitic counterpart, did not mediate lysis by either human or rabbit complement. The expression of a recombinant antibody exhibiting both functional binding site and Fc region capacities shows that the baculovirus system could be employed in the production of therapeutic Ig.

Fibroblast growth factor–binding protein expression changes with disease progression in clinical and experimental human squamous epithelium

Basic fibroblast growth factor (bFGF) is synthesized by a wide variety of normal and malignant cells. However, bFGF cannot exert its effects unless it gets outside of the cell. Since it lacks a signal sequence to direct secretion, the method by which cells release it remains unclear. A 17 kDa secreted binding protein for bFGF (FGF‐BP, HBp‐17) is expressed at high levels in squamous cell carcinoma (SCC) and transformed keratinocytes and may act as a chaperone to transport bFGF outside of the cell. In our study, FGF‐BP mRNA expression in normal keratinocytes was higher than in 5/5 SCCs. Using a new monoclonal antibody, we demonstrate that FGF‐BP can dimerize. Immunoassays demonstrate that normal keratinocytes have a higher level of FGF‐BP than SCCs. In normal human squamous epithelium, we observed diffuse, moderate to intense cytoplasmic and membranous expression of FGF‐BP. Expression decreased and became focal with disease progression to invasive cancer. Injection of immortalized but non‐tumorigenic HaCaT cells transduced with FGF‐BP into normal human skin xenografts failed to result in tumors. Transfection of FGF‐BP into the SCCs Det 562 and FaDu did not promote tumor growth more than controls, and peri‐tumoral microvessel density was lower in FGF‐BP–transfected than in control tumors. Taken together, these data suggest that FGF‐BP expression in squamous epithelium does not play an important role in progression to invasive carcinoma.