Bruce Persky

The influence of ethanol on cell membrane fluidity, migration, and invasion of murine melanoma cells☆

The short-term effects of ethanol (85.4, 170.8, and 256.2 mM) on cellular viability, proliferation, migration, and invasion were investigated on murine melanoma cells. Experiments with the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene indicated that the two highest concentrations of ethanol induced low microviscosity (high lipid fluidity). Cellular viability and proliferation, as determined by the incorporation of [3H]IdUR, were unaffected by all three concentrations of ethanol. A membrane migration assay and a collagen type IV invasion assay evaluated cellular migration and invasion, respectively. For B16F10 and K1735 cells, the migration rate was significantly increased by 170.8 and 256.2 mM concentrations of ethanol. Although the invasion of B16F10 cells was not affected, invasion of K1735 cells was inhibited by 170.8 and 256.2 mM ethanol. The effect of ethanol on the cytoskeleton was monitored by fluorescent staining of F-actin. In contrast to untreated cells, F-actin staining of 256.2 mM ethanol-treated cells showed spike-like projections from the cell surface. Our findings suggest that ethanol can influence cell migration and invasion in vitro, as well as F-actin organization.

The effect of butyric acid and retinoic acid on invasion and experimental metastasis of murine melanoma cells

The effect of butyric acid (BA) and all trans-retinoic acid (RA) on murine melanoma cells was investigated in vitro and in vivo. The in vitro assays included 3H-IdUR incorporation, adhesion, migration and invasion experiments. Butyric acid decreased 3H-IdUR cellular incorporation within 24h and increased adhesion as measured by trypsin release of 3H-IdUR labelled cells from either polycarbonate (p.c.) or Matrigel-coated p.c. membranes. Migration and invasion rates after 72h were quantified by scanning electron microscopy (SEM). The invasion barrier consisted of Matrigel-coated p.c. membranes. Butyric acid significantly enhanced migration and invasion of B16a cells, while RA significantly decreased migration and invasion of B1 6a and K-1735 cells. Subcutaneous administration of either BA or RA pellets significantly decreased the number of lung nodules in the experimental metastatic assay. The experimental metastatic assay is defined as a tail vein inoculation protocol followed by subsequent lung evaluation.

Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

SummaryClonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson’s Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best’s carmine, Page-Green method for inclusion bodies, and Snook’s reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.

Diffusion of dextrans and microspheres in the human amniotic basement membrane model

The human amniotic basement membrane model was utilized to determine diffusion ratios of dextrans and beads according to size selectivity. Diffusion through both intact and denuded amnions was determined after 24 and 72 h. Four neutrally charged fluorescein isothiocyanate labeled dextrans, having molecular weights of 17 900, 42 000, 71 200 and 156 000, diffused through the amnion. The amnion functioned as a sieve in that the passage of dextrans was increasingly restricted as molecular weight increased. In contrast, uncharged latex microspheres (1·05um ± 0·07 µm (SD)) and fluorescent carboxylated microspheres (1·57 µm ± 0·13 um (SD)) failed to pass through the amnion. Light and electron microscopy revealed no preformed channels through which the 1·05 µm microspheres could pass through the amnion. Statistical analysis of cross-sectional thickness of individual and similarly treated amnions (intact or denuded) showed a difference in thickness (P = 0·05).