AN APPROACH TO THE CHARACTERIZATION OF FIBRONECTIN-INTERACTION WITH MAMMALIAN CELLS

Cell adhesion is a fundamentally important process which has been implicated in morphogenesis, metastasis and wound healing. Fibronectin (Fn), a large glycoprotein present in body fluids, the extracellular matrix, and on the cell surface, mediates adhesion of fibroblastic cells. To study the interaction of Fn with Chinese Hamster Cell (CHO) cell membranes, latex beads coated with H('3)-Fn (Fn-beads) were used as surface probes. Binding of Fn-beads was independent of temperature, divalent cations, and metabolic activity. Identification of fibronectin-receptors has been problematical. To study Fn binding components, Fn-beads were pre-incubated with purified glycosaminoglycans (GAGs) and glycolipids. Among the GAGs tested, heparin and heparan sulfate blocked bead binding. Only sialylated glycolipids, GT(,1) and GD(,1) were inhibitory; however, neuraminidase treatment of cells had no effect. It was further shown that Fn-bead binding could be blocked by pre-treating cells with papain. Furthermore, papain digestion releases cellular material which blocks Fn-bead-cell binding. Beads coated with a fragment of Fn which binds to cells but not heparin (F105) were also blocked by soluble papain digests. It was observed that the ability of F105-beads to bind to CHO cells was dependent on surface charge as F105 on uncharged beads did not bind to cells; whereas, F105 on positive or negative beads displayed cell binding activity. The active component in the papain digests was apparently macromolecular (i.e. non-dialysable) and heat stable (i.e. 100(DEGREES)C for 15 min.). This suggested the inhibitory factor is more likely a glycopeptide, rather than a GAG or glycolipid. The findings of this research can be summarized as follows: (1) the expression of cell binding of Fn and Fn fragments can be modulated by the chemical nature of the surface used for adsorption; (2) factors can be released by proteolytic digestion which block Fn and Fn-fragment bead binding; and (3) since bead binding can be done under conditions which reflect initial Fn-cell interaction, it seems likely that the component(s) identified in this way may play a direct role in the recognition phases of cell adhesion to Fn. ^

RUNOFF FROM COAL PILES: EFFECTS ON THE ENVIRONMENT AND THE PUBLIC'S HEALTH

The use of coal for fuel in place of oil and natural gas has been increasing in the United States. Typically, users store their reserves of coal outdoors in large piles and rainfall on the coal creates runoffs which may contain materials hazardous to the environment and the public's health. To study this hazard, rainfall on model coal piles was simulated, using deionized water and four coals of varying sulfur content. The simulated surface runoffs were collected during 9 rainfall simulations spaced 15 days apart. The runoffs were analyzed for 13 standard water quality parameters, extracted with organic solvents and then analyzed with capillary column GC/MS, and the extracts were tested for mutagenicity with the Ames Salmonella microsomal assay and for clastogenicity with Chinese hamster ovary cells.^ The runoffs from the high-sulfur coals and the lignite exhibited extremes of pH (acidity), specific conductance, chemical oxygen demand, and total suspended solids; the low-sulfur coal runoffs did not exhibit these extremes. Without treatment, effluents from these high-sulfur coals and lignite would not comply with federal water quality guidelines.^ Most extracts of the simulated surface runoffs contained at least 10 organic compounds including polycyclic aromatic hydrocarbons, their methyl and ethyl homologs, olefins, paraffins, and some terpenes. The concentrations of these compounds were generally less than 50 (mu)g/l in most extracts.^ Some of the extracts were weakly mutagenic and affected both a DNA-repair proficient and deficient Salmonella strain. The addition of S9 decreased the effect significantly. Extracts of runoffs from the low-sulfur coal were not mutagenic.^ All extracts were clastogenic. Extracts of runoffs from the high-sulfur coals were both clastogenic and cytotoxic; those from the low-sulfur coal and the lignite were less clastogenic and not cytotoxic. Clastogenicity occurred with and without S9 activation. Chromosomal lesions included gaps, breaks and exchanges. These data suggest a relationship between the sulfur content of a coal, its mutagenicity and also its clastogenicity.^ The runoffs from actual coal piles should be investigated for possible genotoxic effects in view of the data presented in this study.^

PHENOTYPIC ASSOCIATION OF GENE AMPLIFICATION WITH MULTIPLE DRUG RESISTANCE IN VINCRISTINE RESISTANT CHINESE HAMSTER OVARY CELLS

Resistance of tumors to pharmacologic agents poses a significant problem in the treatment of human malignancies. This study overviews the scope of clinical resistance and focuses upon current research attempts toward investigation of the phenomenon of multidrug resistance (MDR).^ The objective of this investigation was to determine whether gene amplification had a role in the development of the MDR phenotype in Chinese hamster ovary cells (CHO) primarily selected for resistance to vincristine (VCR). A DNA fragment, previously shown to be amplified in two independently derived Chinese hamster cell lines exhibiting the MDR phenotype, was also amplified in VCR hamster lines. Sequences flanking this fragment were shown to contain coding information for a 4.3 kb transcript overproduced in VCR cells. These sequences were not enriched in double minute DNA preparations isolated from VCR cells. There was an approximately forty-fold increase in both the level of gene amplification and transcript overproduction in the VCR cell lines, independent of the level of primary resistance. This DNA amplification and overproduction of the 4.3 kb transcript was also demonstrated in CHO cells independently selected for resistance to Adriamycin and vinblastine.^ All the DNA sequences of two hamster cDNA clones containing 785 and 932 base pair inserts showed direct homology to the published mouse mdr sequences (about 90%). This sequence conservation held for only portions of the gene when the human mdr1 sequences were compared with those from either the mouse or hamster.^ Somatic cell hybrids, constructed between VCR CHO cells and sensitive murine cells, were used to determine whether there was a functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of MDR-associated amplified sequences, overexpression of the mRNA encoded by these sequences, overexpression of the mRNA encoded by these sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the VCR CHO cells which is responsible for MDR in these cells. ^