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Abnormalities in differentiation are common occurrences in human cancers. Treatment of human melanoma cells with the combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) results in a loss of tumorigenic potential that correlates with an irreversible suppression in proliferative ability and induction of terminal differentiation. It is hypothesized that this is associated with the differential expression of genes that may directly regulate cancer cell growth and differentiation. To define the relevant gene expression changes that correlate with and potentially control these important cellular processes a differentiation induction subtraction hybridization (DISH) scheme is being used. A temporally spaced subtracted differentiation inducer treated (TSS) cDNA library was constructed and differentially expressed DISH clones were isolated and evaluated using a high throughput microchip cDNA (Synteni) array screening approach. Verification of differential gene expression for specific cDNAs was confirmed by Northern blotting. The temporal kinetics of regulation and the expression pattern of DISH genes were also evaluated by microchip cDNA array screening. Using this approach with 1000 DISH cDNA clones (approximately 10% of the DISH library) has resulted in the identification and cloning of both 26 known and 11 novel cDNAs of potential relevance to growth control and terminal differentiation in human melanoma cells.

The importance of angiogenesis in malignant tumor growth has been interpreted mainly in terms of oxygen and nutrient supply. Here we demonstrate its fundamental role for tumor invasion of malignant human keratinocytes in surface transplants on nude mice. Distinct patterns of angiogenesis and vascular endothelial growth factor receptor-2 (VEGFR-2) expression allowed us to distinguish between benign and malignant cells. Functional inactivation of VEGF-R2 by a blocking antibody disrupted ongoing angiogenesis and prevented invasion of malignant cells, without reducing tumor cell proliferation. The reversion of a malignant into a benign phenotype by halting angiogenesis demonstrates a significant function of vascular endothelium for tumor invasion.

Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs) which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75) into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

A clonal cell line (designated MOE CL1) was isolated from the olfactory mucosa of the mouse. A number of growth and biochemical parameters were studied. Our experiments suggest that MOE CL1 cells are olfactory basal cells (undifferentiated neuroblasts).

The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers of 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth of Saccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2, 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminating Candida albicans, Saccharomyces cerevisiae, Aspergillus niger or Fusarium moniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems.

Comparisons have been made of cell surface glycoproteins, concanavalin A agglutinability, and cloning efficiencies in liquid media of ICR 2A (haploid frog cells), ICR 2A M (three cloned populations of haploid frog cells resistant to 5 microgram per ml of the polyene antibiotic mediocidin), and ICR 2A M/MV cells (five cloned populations of morphologically variant haploid frog cells produced by exposure of the parental cells to the combined effects of mediocidin and an RNA mammalian virus, Mengo virus). Independently isolated ICR 2A M/MV clones exhibited altered cell surface glycoproteins, increased concanavalin A agglutinability, and enhanced cloning efficiency in liquid media when compared with ICR 2A parental cells. In contrast, ICR 2A M cells had properties similar to ICR 2A cells, with the exception of the former's increased resistance to mediocidin. The differences in properties between ICR 2A M/MV and ICR 2A cells suggest that alterations resembling transformation have occurred in ICR 2A M/MV cells as a consequence of combined treatment with mediocidin and Mengo virus.