Diane Horn

Regulation of Cell Growth by Recombinant Oncostatin M

Oncostatin M is a novel growth regulator originally isolated from differentiated human histiocytic lymphoma cells and activated T-lymphocytes based on its ability to inhibit the growth of A375 melanoma cells. We report here that oncostatin M is a widely acting regulator which alters the growth and/or morphology of cells derived from a variety of cancer cell types. At picomolar concentrations, recombinant oncostatin M inhibited the growth of 13/24 tumor cell lines. Six out of 7 lung cancer cell lines were inhibited by oncostatin M, but none of 6 colon cancer cell lines were affected. Oncostatin M also stimulated the growth of some normal cells (3/6), indicating that it, like many growth regulators, is bifunctional. Oncostatin M receptors appear necessary but not sufficient for a growth response to oncostatin M, since none of the cell lines lacking receptor responded to oncostatin M, whereas many but not all cell lines with receptor responded to oncostatin M. Receptor size (Mr congruent to 150,000) was similar for cells in which growth was inhibited, stimulated, or unaffected by oncostatin M.

Bromodeoxyuridine substitution in mammalian DNA can both stimulate and inhibit restriction cleavage

Abstract Total replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA causes a 5-fold stimulation in the rate of DNA cleavage by Mbo I. This is the first report of the stimulation of restriction endonuclease activity by 5-bromodeoxyuridine and is in contrast to the inhibition found with other restriction enzymes. We propose a hypothesis to rationalize these results.

Glucocorticoids and melanoma: Receptor properties of dexamethasone sensitive and resistant tumors

We have grown solid tumors using dexamethasone-sensitive (clone 6) and -resistant (clone 5) cells cloned from RPMI 3460 Syrian hamster melanoma. Clone 6 but not clone 5 tumor growth was retarded by dexamethasone, indicating that these tumors retain the growth sensitivity to the hormone characteristic of the cells from which they are derived. Both tumor types contain cytosolic glucocorticoid receptors (significantly higher levels in clone 5 tumors) with similar affinity and steroid specificity characteristics and these can exist as stable, activated (nuclear binding) complexes. Despite these similarities the receptor in the two tumor types differ by some physiochemical criteria. By sucrose gradient analysis, cytosols from both tumors contain 7S receptor complexes but clone 6 contains an additional 13S form. Activated receptors isolated by DEAE-cellulose chromatography from both clone 5 and 6 tumor cytosols sediment as a single peak at 4-5S. However, the DEAE-cellulose profiles indicate that clone 6 but not clone 5 activated complexes (Peak I) appear heterogeneous with respect to charge. Interestingly, DNA-cellulose chromatography indicates that activated receptors from clone 5 tumor cytosols may bind more tightly to DNA than those from clone 6. We are investigating these receptor differences in more detail to determine more precisely the role and pathways of action of glucocorticoid hormones in melanoma.

Clonal variants of a melanoma cell line sensitive to growth inhibition by dexamethasone

Abstract We have isolated and characterized two clones of the RPMI 3460 Syrian hamster melanoma cell line which exhibit different responses to the synthetic glucocorticoid dexamethasone. In the presence of 10 nM dexamethasone, one clone (clone 6) exhibits the growth inhibition, morphological alterations, and reduction in final cell density observed in the parental RPMI 3460 cell line. In contrast, the other clone (clone 5), although exhibiting a reduction in final cell density, fails to exhibit the growth inhibition and morphological alterations. Thus, the effect of dexamethasone on growth and morphology can be expressed separately from the effect of dexamethasone on final cell density in these cells. This observation suggests that the two sets of responses can be controlled separately and that glucocorticoids may exert their influence through different or divergent biological pathways. In vitro receptor assays suggest that the different phenotypes of clone 5 compared with clone 6 cells cannot be explained by an absence of or reduction in cytosolic glucocorticoid receptor, in clone 5 cells. Additional receptor characterization suggests that the different responses to dexamethasone of clone 5 and clone 6 cells do not reflect changes in the ability of receptor to exist in a stably activated form. Differences in the accumulation or depletion of extracellular components in the growth medium also do not seem to be responsible for the altered phenotype of clone 5 vis-a-vis clone 6 cells.

Steroid derivatives for electrophilic affinity labelling of glucocorticoid binding sites: Interaction with the glucocorticoid receptor and biological activity

To investigate the possible use of electrophilic affinity labelling for the characterization of glucocorticoid receptors, different chemically reactive derivatives of deoxycorticosterone (deoxycorticosterone 21-mesylate and deoxycorticosterone 21-(1-imidazole) carboxylate), dexamethasone (dexamethasone 21-mesylate, dexamethasone 21-iodoacetate and dexamethasone 21-bromoacetate) and progesterone (21-chloro progesterone) were tested for their ability to bind irreversibly to the glucocorticoid receptor from goat lactating mammary gland. Using partially purified receptor, only one of the steroids tested, dexamethasone 21-mesylate (DXM-M) was found more effective than dexamethasone (DXM) in preventing exchange of radioactive dexamethasone in the receptor binding site. The affinity of DXM-M for the glucocorticoid receptor, measured by competitive binding assay, was 1/15 that of DXM. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of the [3H]-DXM-M labeled glucocorticoid receptor revealed a specific covalently radiolabeled fraction corresponding to an apparent molecular weight of 75,000 to 80,000. The biological activity of DXM-M was studied in RPMI 3460-clone 6 Syrian hamster melanoma cells, a cell line which is sensitive to growth inhibition by glucocorticoids. Like DXM, DXM-M inhibits the growth of RPMI 3460-clone 6 cells and it acts as a slowly reversible glucocorticoid agonist at concentrations which correlate with the affinity of DXM-M for the glucocorticoid receptor in vitro.