Sandra L. White

Carbohydrate moieties of glycoproteins. A re-evaluation of their function.

Abstract Glycosylation-deficient mutants and inhibitors of glycosulation have been used to investigate the biological function of the carbohydrate moieties of glycoproteins. These and other experimental findings are reviewed and critically evaluated in the present treatise. An hypothesis is proposed to explain the biological significance of the covalent attachment of carbohydrate to protein. It is proposed that the carbohydrate acts as a chemical ‘tag’ which, upon interaction with a specific intracellular membrane receptor, directs glycoproteins to specific cellular organelles following synthesis on the rough endoplasmic reticulum. It is further proposed that secretion of extracellular products is a more general phenomenon which appears not to be absolutely carbohydrate dependent. Also, data are presented which support the view that the carbohydrate moeity is required for the proteolytic or conformational stabilization of the protein component of glycoproteins, but not for the mediation of protein specific biological activity. A model is presented which suggests, for the first time, that the localization function of carbohydrates is not restricted to lysosomal enzymes.

Function of glycoprotein glycans

Abstract Recent results, obtained simultaneously in several laboratories, are reviewed which strengthen earlier notions that the glycan moieties of some glycoproteins play important roles in (1) maintenance of protein conformation and solubility, (2) proteolytic processing and stabilization of the polypeptide against uncontrolled proteolysis, (3) mediation of biological activity, (4) intracellular sorting and externalization of glycoproteins, and (5) embryonic development and differentiation.

Stimulation of DNA synthesis in murine lymphocytes by the drug swainsonine: immunomodulatory properties

Swainsonine, an inhibitor of Golgi alpha-mannosidase II, has recently been shown to have potent antimetastatic activity in experimental metastasis assays. In the case of systemic administration, the possible mechanism of action is unknown; the results reported here indicate that it can be explained at least in part by swainsonine stimulation of lymphocyte proliferation. In the present experiments, the standard in vitro mitogenic stimulation assay was used to test the effect of swainsonine on spleen cells. Treatment of spleen cell cultures with the optimum concentrations of the drug enhanced proliferation by 80-146%, as measured by (3H) thymidine incorporation, relative to untreated cultures. Similarly, when spleen cell cultures were prepared from mice maintained on swainsonine-supplemented drinking water, proliferation was stimulated at least 3-fold relative to cultures derived from animals maintained on regular water. The enhanced mitogenesis is apparently not directly related to increased expression of Concanavalin A (Con A) binding sites, since swainsonine induced mitogenesis is not inhibited by alpha-methyl-mannoside in contrast to Con A induced mitogenesis which is completely inhibited. These results suggest that the antimetastatic effect of systemically administered swainsonine is at least in part related to its ability to enhance proliferation of those specific cell populations involved in immune surveillance. This represents the first demonstration of a mechanism of action of swainsonine on the immune system.

Carbonoyloxy analogs of the anti-metastatic drug swainsonine: Activation in tumor cells by esterases

Swainsonine (SW), a plant alkaloid and inhibitor of alpha-mannosidases, has been shown to inhibit N-linked oligosaccharide processing and to block tumor cell metastasis in mice. In this study, a series of SW analogs were chemically synthesized and compared for inhibition of complex-type N-linked oligosaccharide processing in cultured MDAY-D2 tumor cells, for inhibition of alpha-mannosidases in vitro, and for stimulation of bone marrow proliferation in vivo. Carbonoyloxy substitutions at the 2 and 8 carbons of SW reduced inhibitor activity by 2-3 orders of magnitude for Jack Bean and MDAY-D2 tumor cell lysosomal alpha-mannosidases in vitro. However, 2-p-nitrobenzoyloxy-, 2-octanoyloxy- and 2-butanoyloxy-derivatives of SW retained full activity as inhibitors of Golgi oligosaccharide processing in viable MDAY-D2 tumor cells. Inhibition of oligosaccharide processing was reduced by the esterase inhibitor diethyl p-nitrophenyl phosphate, suggesting that although 2-p-nitrobenzoyloxy-SW, 2-octanoyloxy-SW and 2-butanoyloxy-SW are relatively poor inhibitors of alpha-mannosidases in vitro, the compounds enter cells at a rate comparable to that of SW, and are converted to SW by cellular esterases. The more lipophilic esters, 2-benzoyloxy-SW, 2-toluoyloxy-SW, 8-palmitoyloxy-SW and 8-myristinoyloxy-SW, showed IC50 values at least 10 times higher for inhibition of Golgi oligosaccharide processing, probably due to less efficient entry of the compounds into tumor cells. The anti-metastatic activities of SW and two analogs were tested and shown to correlate with the IC50 values for inhibition of Golgi oligosaccharide processing in cultured tumor cells. In vivo, SW and the analogs were administered intraperitoneally to mice and found to have comparable activities as stimulators of bone marrow cell proliferation. Carbonoyloxy substitutions at the 2- or 8-position of SW with other chemical groups may lead to new drugs with improved pharmacokinetics and anti-cancer activity.

An assessment of the effects of swainsonine on survival of mice injected with B16-F10 melanoma cells

Systemic administration of swainsonine, an indolizidine alkaloid, inhibits the experimental metastasis of B16-F10 murine melanoma cells. This activity can be attributed primarily to swainsonine-mediated enhancement of host natural killer cell activity. As one next step towards investigating the potential therapeutic utility of this drug, its efficacy in enhancing host survival in the same B16-F10 model system has been assessed. In studies employing intravenously injected tumor cells, pretreatment of mice with swainsonine-containing drinking water provided a reproducible protective effect for the host. This prolongation of survival was substantially enhanced when swainsonine was administered in combination with either of two other immunomodulators, polyinosinic : cytidylic acid (poly-IC) or interleukin-2. In studies in which combinations of these agents were administered after intravenous injection of tumor cells, or after subcutaneous implantation, a greatly reduced effect on host survival was observed. However, when used in combination with cyclophosphamide (to block the effects of suppressor T cells), swainsonine did increase mean survival time. The implications of these results for the use of swainsonine in treatment of metastatic or localized disease, together with its potential mechanism(s) of action, are discussed.

Induction of Macrophage Tumoricidal Activity, Major Histocompatibility Complex Class II Antigen (Iak) Expression, and Interleukin-1 Production by Swainsonine

Previous studies in our laboratory have shown that the reported antitumor activity of systemically administered swainsonine, an indolizidine alkaloid, is due at least in part to immune modulation involving effector cells (Humphries, M.J.; Matsumoto, K; White, S.L.; Olden, K. Cancer Res. 48:1410-1415; 1988 and White, S. L.; Schweitzer, K.; Humphries, M.J.; Olden, K. Biochem. Biophys. Res. Commun. 150:615-625; 1988). In this report, studies are presented to show that swainsonine was effective in activating peritoneal macrophages to cytotoxicity against tumor cells. Stimulation of tumoricidal activity of macrophages was associated with increased secretion of interleukin-1 (IL-1) and expression of the Iak major histocompatibility complex (MHC) antigen on the cell surface. The 3-fold stimulation of cytotoxicity observed in these in vivo studies was comparable to that obtained with Corynebacterium parvum, a commonly used in vivo activating agent. The in vitro incubation of thioglycollate-elicited peritoneal macrophages with swainsonine consistently resulted in levels of activation (6- to 8-fold) comparable to that obtained by treatment with known in vitro macrophage activating agents such as lipopolysaccharide (LPS) or recombinant gamma-interferon (rIFN-gamma). The stimulation observed by using swainsonine in combination with LPS was additive, suggesting different mechanisms of action. These studies have important implications not only for treatment of cancer, infectious diseases, and immune suppressive disorders, but also for elucidation of the mechanism of macrophage activation.

Swainsonine stimulation of the proliferation and colony forming activity of murine bone marrow

Swainsonine, an indolizidine alkaloid, was recently reported to exhibit both antineoplastic and immunomodulatory activities (Humphries, M.J.; Olden, K. Asparagine-linked oligosaccharides and tumor metastasis. Pharmacol. Ther. 44:85-105; 1989). In this study, we show that systemically administered swainsonine promoted the proliferation of murine bone marrow (BM) cells. Animals that received swainsonine intravenously exhibited a significant increase (approximately 5-10 fold) in BM cellularity, engraftment efficiency, and colony forming unit activity using in vitro or in vivo assays. BM cells derived from swainsonine-treated animals or treated with swainsonine in vitro also exhibited a 4-5 fold increase in [3H]-thymidine incorporation, suggesting that a larger fraction of the cells was in the S-phase of the cell cycle. This provides the first evidence that swainsonine, which stimulates the production of cytokines by cells of the immune system, promoted the proliferation of BM progenitor cells. These results suggest that swainsonine could prove valuable in patients undergoing intensive chemoradiotherapy or autologous BM transplantation by decreasing or possibly eliminating leukopenia or myelosuppression often associated with these procedures; it may also be a useful probe to investigate the mechanism of normal hematopoieses.

Swainsonine modulation of protein kinase C activity in murine peritoneal macrophages.

The level of Ca2+, phospholipid-dependent, protein kinase C (PKC) activity in murine peritoneal macrophages treated with swainsonine, an indolizidine alkaloid, has been investigated. The present studies are based on our recent report that murine peritoneal macrophages are activated by swainsonine (Grzegorzewski, K.; Newton, S.A.; Akiyama, S.K.; Sharrow, S.; Olden, K.; White, S.L., Cancer Commun. 1:373-379, 1989). Presently, we have demonstrated that macrophages treated with swainsonine exhibited a substantial increase in PKC activity. The activity was enhanced as much as 4- to 5-fold over that obtained in untreated macrophages and was inhibited by H-7 (1-[5-isoquinoline sulphonyl]-2-methylpiperazine), D-sphingosine, or a monoclonal antibody specific for the active site of PKC. This represents the first report to demonstrate an effect of swainsonine on a second messenger system known to be involved in tumor promotion and macrophage activation. Elevation of PKC activity occurred much more slowly in swainsonine-treated cells than in cells treated with agents known to activate PKC directly, e.g., PMA (4-beta-phorbol-12-beta-myristate-13-gamma-acetate) or gamma-interferon (IFN-gamma). Furthermore the increase in PKC activity was inhibited by alpha-amanitine and cycloheximide, inhibitors of RNA and protein synthesis, respectively. These results suggest that swainsonine enhancement of PKC activity occurred by an indirect and possibly protein-synthesis-dependent mechanism. Whatever its precise mechanism of action, swainsonine provides a potentially important new probe to evaluate PKC mediated events. Selective enhancement of PKC activity may be important not only in elucidating the role of PKC in tumor promotion or macrophage activation but, also, in contributing to development of therapeutic regimens.

Use of antiadhesive peptide and swainsonine to inhibit metastasis.

Metastasis, the spread of tumor cells from a primary to a secondary site within the body, is the principal cause of morbidity and death in cancer patients. Consequently, there is great interest in understanding the biology and biochemistry of the metastatic process and in developing therapeutic strategies for the prevention and/or elimination of metastatic lesions. The process of tumor metastasis comprises a sequential series of migratory and invasive events that necessitate the passage of cells through both connective and vascular environments.I4 Thus, malignant cells must possess a phenotype that allows them to perform a multitude of functions. In this chapter, we will review our preclinical findings which suggest that the employment of antimetastatic agents, developed on the basis of knowledge of the cell biology and biochemistry of the metastatic process, may be clinically efficacious in the management of human rnalignancie~.~-”

Lung Cancer in Black Populations of the United States: Overview and Update

In 1986 in the United States, 100,000 men and 49,000 women were expected to develop lung cancer according to an American Cancer Society (ACS) (1986) estimate. This represents about 15% of all cancer cases (22% in men and 11% in women); the incidence of lung cancer has been increasing at up to 10% per year since the 1930s (Cutler and Devesa 1973; Devesa and Silverman 1978). Among the major ethnic groups, Blacks have the highest overall incidence of lung cancer and American Indians the lowest. A recent report (Horm and Kessler 1986) indicated that the incidence of lung cancer in White men had dropped for the first time, from 82.7 per 100,000 in 1982 to 79.3 in 1983. For Black men, the incidence appeared to have increased slightly in this same period (Table 1). In 1983 the incidence for Black men was 125.3, 58% higher than that for White men. The incidence of lung cancer in both White and Black women had not shown any change during the previous decade, and Blacks had higher age-specific rates than Whites (National Cancer Institute [NCI] 1985).