Marvin D. Bregman

Synthesis and isolation of a glucagon antagonist

The glucagon binding to hepatocytes results in an immediate increase in intracellular cyclic AMP [ 11, which is followed by an increase in glycogenolysis and gluconeogenesis [2]. It has been suggested that glucagon may be involved in the pathogenesis of diabetes mellitus [3]. A specific glucagon antagonist could prove useful in determining what relationship the hormone has with this disease state. Previous investigators [4] have provided evidence that ‘(desHis’ )-glucagon’, synthesized by the one-step Edman procedure is a competitive inhibitor of glucagon action on liver plasma membrane adenylate cyclase. A highly purified (des-His’ )-glucagon was synthesized by the solid state Edman procedure, which insures the integrity of the e-amino group, and was shown [5] to be a partial agonist. Therefore, the identity of the antagonist remains in doubt. In view of the potential usefulness of this inhibitor we decided to reinvestigate the reaction of phenylisothiocyanate with glucagon. Here we describe the purification and identity of the glucagon antagonist synthesized by the one-step Edman degradation and report on some of its properties which explain the previous differences.

Prostaglandin A1 and E1 inhibit the plating efficiency and proliferation of murine melanoma cells (Cloudman S-91) in soft agar.

PGA1 and PGE1 reduced the plating efficiency and inhibited proliferation of Cloudman S-91 murine melanoma cells in a dose dependent manner, as assessed by their effects on colony formation in soft agar. PGF2α did not reduce plating efficiency but was as effective as PGA1 in raising cAMP and cGMP levels. This data suggests that the inhibition of Cloudman S-91 murine melanoma cell growth occurs via a non-cyclic nucleotide mechanism.

Anchorage-independent growth of murine melanoma in serum-less media is dependent on insulin or melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (MSH) is known to stimulate melanogenesis in murine melanoma, particularly in Cloudman S-91 melanoma cells. The effects of MSH and insulin on the proliferation of S91 murine melanoma cells have aroused controversy; in various reports, both hormones have been reported to either stimulate or inhibit murine melanoma growth. In our studies both MSH and insulin stimulated the colony-forming ability and the proliferative capacity of S-91 murine melanoma cells grown in soft agar with either serum-supplemented or serum-less medium. Unless insulin and/or MSH were present, Cloudman S-91 melanoma cells failed to clone in soft agar. The insulin effect was greater than that of MSH, and was more pronounced in serum-less than in serum-supplemented medium. The concurrent treatment of S91 melanoma cells with both MSH and insulin resulted in a greater increase in the total number of colonies formed than caused by treatment with either hormone alone. The combined MSH-insulin stimulation of anchorage-independent growth was specific, since the effect could not be mimicked by epidermal growth factor (EGF), gonadotropin-releasing hormone (GRH), luteinizing hormone (LH), nerve growth factor (NGF) or platelet-derived growth factor (PDGF). Therefore, MSH and insulin may be specific growth factors for murine melanoma cells.

In vitro modulation of human and murine melanoma growth by prostanoid analogues.

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16,16-dimethyl PGA1, 16,16-dimethyl-PGA2, 16,16-dimethyl-18-oxa-PGA2 and trans-delta-2-15-alpha acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE1-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD2 (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA1 and PGE1 were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD2 was the most active prostaglandin, 2-3 times more potent than PGA1; PGE1 was a very weak inhibitor.

Iodination associated inactivation of β-melanocyte stimulating hormone

Abstract Contrary to other published reports, iodinated β-melanocyte stimulating hormone (β-MSH) is without biological activity as measured by frog skin bioassay, melanoma (mouse S-91) adenylate cyclase assay, or melanoma tyrosinase assay. Inactivation results in part from oxidation of the methionine residue by the chloramine T and sodium metabisulfite used in the iodination reaction. Replacement of the methionine of β-MSH with norleucine by solid phase synthesis results in an analogue which is more resistant, but not completely resistant, to inactivation. Thus, in order to obtain a biologically active radioligand for radioreceptor studies, further tailoring of the hormone and/or modification of the iodination procedure will be needed.

Long-term and residual melanotropin-stimulated tyrosinase activity in S91 melanoma cells is density dependent.

SummaryCell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer culture. Continuous exposure of melanoma cells to α-melanotropin or its potent analog [Nle4,D-Phe7]-α-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins were subcultured to an initial low cell density and maintained in contact with α-MSH or [Nle4,D-Phe7]-α-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at initial densities ranging from 0.2 to 3.2×106 cells/flask, and exposed for 24 h to 10−7M α-MSH, only the cultures seeded at low densities (0.2 and 0.4×106 cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure to melanotropin. Other flasks of various cell densities were treated with 10−7 M α-MSH or [Nle4,D-Phe7]-α-MSH for 24 h, followed byremoval of the melanotropins from the culture medium. The magnitude and duration of theresidual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density. These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins and express increased tyrosinase activity.

(Des-Histidine1) (Nϵ-phenylthiocarbamoyllysine12)-glucagon: Effects on glycogenolysis in perfused rat liver

Abstract (Des-Histidine 1 ) (N ϵ -phenylthiocarbamoyllysine 12 )-glucagon, synthesized by the one-step Edman degradation procedure is a competitive inhibitor of glucagon action in the rat liver plasma membrane adenylate cyclase system. However, in the perfused rat liver, the compound did not inhibit glucagon stimulated glycogenolysis even when used at a concentration 100-fold in excess of native glucagon. Instead, it showed a weak potency, but full agonist activity, stimulating liver glycogenolysis to 100% of the level obtained by glucagon. These results are discussed in terms of the possible mechanism(s) of glucagon action.

Adenosine and divalent cation effects on s-91 melanoma adenylate cyclase.

Abstract Adenosine inhibits S-91 melanoma membrane adenylate cyclase. This inhibition is observed with all activators tested; α-melanocyte-stimulating hormone, [norleucine 4 ]α-melanocyte-stimulating hormone, prostaglandin-E 2 , and fluoride ion. This inhibition is also observed with the ribose modified adenosine analog adenine-9-β- d -arabinofuranoside. However, “R” site (stringent for the ribose moiety) analogs such as 2-chloroadenosine and 6-methyladenosine were without effect. Addition of manganous ion (Mn 2+ ), a potent activator of adenylate cyclase, markedly enhances the inhibition by adenosine. The nucleoside specificity, Mn 2+ requirement, and lack of reversal by the potent R-site antagonist, 3-isobutyl-1-methylxanthine, suggest that the S-91 melanoma membrane adenylate cyclase system contains only P-type (stringent for purine moiety) receptor sites.

Effect of tumor colony definition on ionizing radiation survival curves of melanoma-colony forming cells.

Definition of survival and measurement of colony size in soft agar assays is important in establishing in vitro radiation survival curves. Conventionally, survival is assessed according to colony-forming ability. The distinction between small colonies that are abortive and those that are viable often involves a difficult and arbitrary choice for the investigator. We have examined the effect of different minimum colony sizes (greater than or equal to 25, greater than or equal to 50, greater than or equal to 75, and greater than or equal to 100 cells) on ionizing radiation survival curves for cells from established murine (CCL 53.1) and human (M1RW5) melanoma cell lines as well as from short-term human melanoma cell strains (C8146A, C8146C, C8161, C83-2C, C82-7A1, and C8442) and patient biopsy (83-4). Single cell suspensions were plated in the upper layer of the agar bilayer and cells were irradiated by single dose X rays. Giant cells did not form in colonies containing 50 or more cells. D0 values were highest (D0 values, from 390 to 100 cGy) for cells forming smaller colonies (greater than or equal to 25 cells, greater than or equal to 4-5 doublings) and lowest (D0 values, from 190 to 50 cGy) for cells forming larger colonies (greater than or equal to 100 cells, greater than or equal to 6-7 doublings). Therefore, apparent radiosensitivity was dependent on colony size selected for analysis. Precise measurement of colony size was important in establishing radiation survival curves because errors in determining the colony size will alter apparent radiosensitivity of cells. These results should help define the biological meaning of tumor colony growth in semisolid medium, and alter the interpretation of survival curves which measure sensitivity to agents using this assay.

Glucagon structure-function relationships using isolated rat hepatocytes.

The ability of glucagon and several of its semi-synthetic analogues to stimulate glucose production in isolated rat hepatocytes was measured and compared for relative potencies. The order of decreasing biological activities of glucagon in this assay was as follows: glucagon greater than [HArg12]-glucagon greater than [des-Asn28, Thr29][homoserinehydrazide27]-glucagon approx. equal to [des-His1]-glucagon greater than [des-Asn28, Thr29][homoserinelactone27]-glucagon greater than [des-Asn28, Thr29]-[n-butylhomoserineamide27]-glucagon greater than glucagon1-21. Qualitatively, these results are similar to those obtained previously in the hepatic plasma membrane adenylate cyclase assay. Minor exceptions were noted for the hydrazide derivative and the partial agonist [des-His1]-glucagon, both of which were slightly more potent relative to glucagon in the glycogenolytic assay than in the adenylate cyclase assay. The assay provides important insight into glucagon structure-function relationships.