Carcinogenesis in human skin grafted to SCID mice.

To directly examine a multistage carcinogenesis model and the role of UV light in human tissues, we grafted human skin onto mice with severe combined immunodeficiency disease. We found that the maximum dose of UV radiation in the B range (UVB; 280-320 nm) tolerated by these grafts was 500 J/m2 three times weekly. One hundred fifty-one grafted mice were then randomized and observed for a median of 9 months in five groups: no treatment, chemical initiation alone, UVB as a complete carcinogen, initiation plus UVB promotion, and initiation plus UVB and phorbol ester promotion.

Cultivation of normal human epidermal melanocytes.

An important approach in studies of normal, diseased, and malignant cells is their growth in culture. The isolation and subsequent culture of human eplderma1 melanocytes has been attempted since 1957 (1-5), but only since 1982 have pure normal human melanocyte cultures been reproducibly established to yield cells in sufficient quantity for biological, biochemical, and molecular analyses (6). Selective growth of melanocytes, which comprise only 3-7% of epldermal cells in normal human skin, was achieved by suppressing the growth of keratinocytes and fibroblasts in epidermal cell suspensions with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the intracellular cyclic adenosine 3', 5' monophosphate (cAMP) enhancer cholera toxin, respectively, which both also act as melanocyte growth promoters.

Mitogenic activity of laminin on human melanoma and melanocytes: different signal requirements and role of beta 1 integrins.

The possible mitogenic activity of laminin (LN) on normal and neoplastic cells of the melanocyte lineage was tested by culturing growth-arrested human melanoma cells and neonatal foreskin melanocytes on LN. Serum-deprived, quiescent melanoma cells proliferated, in serum-free medium, in a dose-dependent fashion to immobilized LN as determined by [3H]thymidine incorporation, cell cycle analysis, and change in cell number. The mitogenic activity of LN on melanoma cells was not mediated through autocrine release of growth factors and was observed with primary or metastatic melanoma cells and with clones isolated from the same metastasis but only on cells expressing very late antigen (VLA)-3 and VLA-6 laminin receptors.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative.

The melanoma differentiation-associated gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21, is differentially expressed during growth, differentiation and progression in human melanoma cells.

The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) induces terminal differentiation with an irreversible loss of proliferative capacity in human melanoma cells. Using subtraction hybridization, cDNAs were identified that display enhanced expression in terminally differentiated and growth arrested human melanoma cells (Jiang and Fisher, 1993; Jiang et al., 1994a).

Experimental local therapy of human melanoma with lytic magainin peptides.

Magainin peptides and model amphipathic peptides exhibit antibiotic activity and are also cytolytic for transformed human cells. Here we demonstrate in vitro that MSI-511 (an all-D amino-acid model magainin peptide) and MSI-130 (a margainin analogue) were more lytic for 17 human melanomas than for normal melanocytes. Melanomas established s.

Abnormal protein tyrosine kinase gene expression during melanoma progression and metastasis.

Protein tyrosine kinases have been implicated in tumor initiation and progression. Here we used Northern blotting to study expression of their genes in cultured normal melanocytes and 19 melanoma cell lines from different stages of tumor progression. We detected transcripts for 2 cytoplasmic (ABL and FES) and 6 receptor (ECK, ERB-B2, FGF-R4, IGFI-R, KDR and TIE) kinases but not for receptors RET or TRK-A.

Regulation of Mel-CAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.

The cell-cell adhesion receptor, Mel-CAM/MUC18, is highly expressed on metastatic melanoma cells and is also detectable on primary melanomas but not on normal melanocytes. Previous studies have shown that increased Mel-CAM/MUC18 expression correlates with tumor thickness and metastatic potential. We show here that normal melanocytes and nevus cells in culture express Mel-CAM/MUC18, but expression is down-regulated when cells are co-cultured with keratinocytes.

Protein kinases in normal and transformed melanocytes.

Aberrant function of protein kinases has been implicated in the development of melanoma. In an effort to define the molecular events involved in initiation and progression of this malignancy, we used RT-PCR to identify protein kinases in both normal and transformed melanocytes. Collectively, we identified seven clones corresponding to previously characterized protein kinases (JAK-1, TYK02, AXL/UFO, IGF1-R, KDR and FER) as well as the recently identified MLK-3/PTK1 protein kinase.

Cellular pathways leading to melanoma differentiation: therapeutic implications.

The induction of differentiation, as evidenced by benign growth characteristics, dendritic morphology, pigmentation capability, and a mature antigenic phenotype, is an attractive theoretical basis for therapy in human melanoma. Melanoma differentiation can be experimentally induced by modulating intracellular pathways involving protein kinase C, tyrosine kinases, and protein kinase A, or by modulating nuclear transcription with retinoids, DNA-damaging agents, and chemotherapeutic drugs. Other experimental differentiating agents include the amino acid tyrosine, histamine receptor antagonists, polyamine antagonists, dimethylsulphoxide, caffeic acid ester, and butyrate.

Biologic and therapeutic significance of MYB expression in human melanoma.

We investigated the therapeutic potential of employing antisense oligodeoxynucleotides to target the disruption of MYB, a gene which has been postulated to play a pathogenetic role in cutaneous melanoma. We found that MYB was expressed at low levels in several human melanoma cell lines. Also, growth of representative lines in vitro was inhibited in a dose- and sequence-dependent manner by targeting the MYB gene with unmodified or phosphorothioate-modified antisense oligodeoxynucleotides.

Isolation and functional characterization of the A32 melanoma-associated antigen.

Cell surface melanoma-associated antigens can mediate cell-cell or cell-substrate adhesion, signal transduction, proteolysis, or immune recognition and play a key role in determining invasive and metastatic competence of the tumor cells. The melanoma-associated antigen, A32, was defined by a murine monoclonal antibody and was immunoprecipitated as a single 113 kDa integral membrane glycoprotein containing sialic acid and HNK-1 carbohydrate moieties. Immunohistochemistry revealed the presence of A32 antigen on most melanomas and nevi but not on normal epidermal melanocytes.

Isolation and analysis of novel human melanocyte-specific cDNA clones.

Human melanoma represents a neurocrest-derived malignancy, occurring in 10% of cases within the setting of a familial predisposition and in 90% of cases with sporadic onset. It is estimated that more than 85% of familial melanomas and about 40% of sporadic melanomas arise from melanocytic precursor lesions. In an attempt to isolate genes that may play a role in human melanocytic progression, we applied subtractive cDNA hybridization, involving repeated rounds of subtraction.

Interactions of melanocytes and melanoma cells with the microenvironment.

Normal or malignant melanocytes interact with the microenvironment through the release of soluble factors from cells and through direct cell-cell contact. Melanoma cells produce a large number of different growth factors and cytokines that affect angiogenesis, stroma formation, motility, and the inflammatory and immune response. Most of the angiogenic growth factors produced by melanoma cells are also mitogenic for fibroblasts.

Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2'-deoxycytidine.

MAGE-1 is a gene that encodes an antigen on a melanoma cell line that is recognized by cytolytic T-cells. We have used a reverse transcription-polymerase chain reaction assay to analyze expression of the MAGE-1 gene by cell lines from different types of tumors, melanomas from different stages of disease progression, normal diploid cell lines, and melanocyte and nevus tissue from which malignant melanomas are derived. MAGE-1 is expressed by melanoma tissue from all stages of disease, but not melanocytes, nevus tissue, or any normal diploid cell line tested.

Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes.

Human tumors can constitutively express cytokines and growth factors, but the extent of this expression has not been investigated. Using 44 different probes to cytokines, growth factors, and their receptors, we tested 21 melanoma and 5 melanocyte cultures for RNA transcript expression by reverse transcriptase-polymerase chain reaction. With 30 amplification cycles, expression of the cytokines interleukin (IL)-1 beta, IL-6, leukemia inhibitory factor (LIF), IL-7, gro alpha, IL-8 and the p35 chain of IL-12 was detected in more than 60% of melanomas.

Adhesion receptors in human melanoma progression.

Melanoma often develops from clinically and histologically well-defined precursor lesions. During progression of normal melanocytes to benign nevi, dramatic changes in the expression of adhesion receptors are observed, most notably loss of E-cadherin which mediates adhesion of melanocytes to keratinocytes, and gain of Mel-CAM which predominantly mediates heterotypic adhesion between cells. Major changes in adhesion receptors also occur when cells progress from dysplastic nevi or biologically early radial-growth-phase primary melanomas to biologically late (tumorigenic) vertical-growth-phase primary melanomas.

Autocrine and paracrine roles for growth factors in melanoma.

Distinct biologic and histopathological features characterizing each stage of tumor progression toward a more aggressive phenotype have been defined in the human melanocytic cell system. One of the most significant aspects accompanying melanoma progression is the acquisition of growth autonomy and the expression of multiple growth factors and receptors by tumor cells but not by normal melanocytes. Among the growth factors produced by melanoma cells, bFGF, TGF-alpha, TGF-beta, PDGF A and B chains, MGSA, and interleukins have been extensively characterized.

Novel and known protein tyrosine kinases and their abnormal expression in human melanoma.

We have used the polymerase chain reaction and Northern blotting to identify protein tyrosine kinases that may play an important role in the process of melanoma initiation and progression. Degenerate primers from the conserved catalytic domain of tyrosine kinase genes were used to amplify and clone partial cDNA sequences from a human melanoma cell line (DX3-LT5.1) and normal human melanocytes.

Development of pemphigus vulgaris-like lesions in severe combined immunodeficiency disease mice reconstituted with lymphocytes from patients.

Pemphigus vulgaris is an autoimmune blistering disease that is induced by binding of antibodies to a 130/85-kD protein complex on epidermal keratinocytes. An in vivo experimental model of this disease was developed by reconstituting severe combined immunodeficiency (SCID) mice with 1-10 x 10(7) PBL from patients with naturally occurring pemphigus vulgaris. Of 49 reconstituted mice, 34 (69%) produced human IgG levels of > 0.

Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.

Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry.

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing.

Expression of colorectal carcinoma-associated antigens in colonic polyps.

Immunohistologic techniques were used to study the expression of colorectal carcinoma-associated antigens in colonic polyps and to compare this with expression in the normal colonic epithelium. Forty-nine polyps were studied using monoclonal antibodies to 16 different blood group and differentiation antigens and carcinoembryonic antigen epitopes. With the Lewis(a) antigen and the two epitopes of CEA recognized by 3D6 and COL-4 expression in polyp tissue was the same as that in the normal colon.

Undifferentiated keratinocytes control growth, morphology, and antigen expression of normal melanocytes through cell-cell contact.

Background: Melanocytes in the normal human epidermis are generally dendritic and neither proliferate nor express melanoma-associated antigens. In culture, on the other hand, melanocytes are bi- to tripolar, proliferate with 2 to 4 day doubling times, and express melanoma-associated antigens. This observation prompted us to investigate the regulatory role of keratinocytes for growth, morphology, and antigen expression of melanocytes.

Growth and invasion of human melanomas in human skin grafted to immunodeficient mice.

An orthotopic model of human melanoma was developed in which malignant cells were injected into human skin grafted to nude and SCID mice. Melanoma cells proliferated and invaded the human skin grafts with characteristic patterns. Three of six melanomas grew as multiple nodules and infiltered the grafts without major architectural changes in the dermis, whereas the others invaded the dermis along collagen fibers with prominent endothelial vessels.