Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

Identification of beta-lymphotoxin as the predominant molecular class of in vitro and in vivo Syrian hamster lymphotoxin

The molecular class of Golden Syrian hamster lymphotoxin produced in vitro and in vivo was determined by size-exclusion high-performance liquid chromatography using silica-based protein separation columns eluted with a 0.1 M sodium phosphate, pH 7.4 buffer containing 0.1% Mr 4000 polyethylene glycol. Lymphotoxin cytolytic activity was quantitated in the column effluent by measuring the ability of the fractions to lyse alpha-L929 cells as indicated by [3H]TdR release. Lymphotoxin activity induced by an 8- or 24-hr or 5-day phytohemagglutinin stimulation of peritoneal leukocytes, by 24-hr phytohemagglutinin-coated alpha-L929-cell stimulation of peritoneal leukocytes, or by 24-hr phytohemagglutinin stimulation of spleen cells occurred in the Mr range of 20,000-56,000, with major components in the 35,000-50,000 beta-lymphotoxin region. No activity was present in the complex (greater than 200,000) region and only minimal activity was detectable in the alpha (70,000-160,000) and gamma (12,000-20,000) regions. In vivo-induced lymphotoxin, obtained by peritoneal lavage 48 hr after intraperitoneal administration of phytohemagglutinin, was entirely beta-lymphotoxin and was not detectable in the plasma. Lymphotoxin produced in vitro and injected simultaneously with the gamma-emitting radionuclide 99mtechnetium, inhibited in vivo development of radiation-induced transplacental carcinogenesis. Thus, Syrian hamster lymphotoxin with antitumor activity consists of glycoproteins with isoelectric points of 4.8-5.2, Mr of 20,000-56,000, and major in vitro and in vivo forms in the beta-lymphotoxin range.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.     

Modulation by all-trans retinoic acid of glycoprotein glycosylation in murine melanoma cells: enhancement of fucosyl- and galactosyltransferase activities

beta-All-trans retinoic acid (RA) treatment of murine S91-C2 melanoma cells decreases in vitro growth and modulates the glycosylation of specific cellular and cell-surface glycoproteins. The effect of RA treatment on [3H]fucose, [3H]galactose, and [3H]glucosamine incorporation was investigated by metabolic labeling followed by analysis of labeled cellular glycoproteins using polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and fluorography. RA treatment dramatically increased the incorporation of the labeled monosaccharides into one glycoprotein of Mr 160,000 (gp160), which has been previously implicated in the growth-inhibitory effect of RA on these cells. Following RA treatment, cell-surface sialic acid residues on gp160 were also more intensely labeled by NaIO4 oxidation and subsequent NaB[3H]4 reduction than were those on gp160 of untreated cells. The activities of fucosyl- and galactosyltransferase increased about 1.5 to 1.9 times after RA treatment. These results suggest that the increased activities of the two glycosyltransferases is responsible for the increased incorporation of fucose and galactose into gp160.     

Recombinant human lymphotoxin effects on osteoblastic cells

Lymphotoxin, or tumor necrosis factor beta, has been shown to be a potent bone resorbing cytokine. In the present study, the effect of recombinant human lymphotoxin on osteoblastic cell proliferation and prostaglandin synthesis was investigated. Lymphotoxin (10(-10)-10(-7) M) caused a significant, dose-dependent decrease of rat osteoblastic cell proliferation. This appeared to be an indirect, prostaglandin-dependent action, since in the presence of indomethacin (1 microM) the lymphotoxin effect was reversed. Subsequently, prostaglandin E2 and prostacyclin (assayed as 6-keto-prostaglandin F1 alpha) levels produced by the osteoblastic cells in response to lymphotoxin were measured. The cytokine caused a dose-dependent increase of these arachidonic acid metabolites, with the maximum effect at 10(-8) M. These results suggest that lymphotoxin's mechanism of action on bone may involve increases in arachidonic acid metabolite synthesis and an indirect, prostanoid-mediated decrease in the proliferation rate of osteoblastic cells.     

An Impaired Uptake of Glucosamine in Tunicamycin Resistant Chinese Hamster Ovary Cells

Tunicamycin resistant mutants (TM^R) were isolated and characterized from Chinese hamster ovary cells. One feature of this TM^R mutants was a marked decrease in incorporation of radioactive glucosamine, both into membrane glycoproteins and G protein of vesicular stomatitis virus.

The cellular uptake and incorporation into acid insoluble materials of various radioactive substances, including glucosamine, galactosamine, mannose, 2-deoxyglucose and leucine, was examined for the purpose of determination whether the reduced incorporation of radioactive glucosamine into glycoproteins was due to a defect in the glycosylation step or decreased uptake of glucosamine by cells.

While incorporation of glucosamine and 2-deoxyglucose into acid insoluble fractions was reduced strikingly in the mutants, the incorporation of mannose and leucine were the same as in the parent cells.

The uptake of glucosamine in TM^R cells was lower than that in the wild type cells, and the Km value for glucosamine uptake differed between the mutants and wild type cells. There was no obvious difference in the uptake of 2-deoxyglucose and mannose.     

Comparison of the cytostatic and cytolytic activity of tumor necrosis factor-alpha and interleukin 1 alpha in human malignant cell lines.

The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 alpha (IL-1 alpha) share many properties, including in-vitro cytotoxicity. Because cytotoxicity can result from either cytolytic or cytostatic activity, and because differentiating between these activities may have clinical relevance, we determined the cytostatic and cytolytic activity of TNF-alpha and IL-1 alpha for the human cell lines ME-180, SiHa (cervical carcinomas) and A375 (melanoma). Results of these analyses showed that IL-1 alpha mediated cytostatic activity only for A375 cells. IL-1 alpha was not cytolytic in the presence or absence of protein synthesis inhibitors. TNF-alpha was cytostatic for A375 and ME-180 cells, and although TNF-alpha was not cytolytic in the absence of protein synthesis inhibitors, it was cytolytic in the presence of protein synthesis inhibitors. These results suggest that the difference between the cytolytic and cytostatic activities of IL-1 alpha and TNF-alpha may have therapeutic implications for the use of these biological response modifiers in the treatment of gynecological malignancies.     

The effect of antilymphocyte globulin (ALG) on human lymphocytes in vitro: inhibition of lymphotoxin production as a sensitive indicator for ALG activity.

The effect of antilymphocyte globulin (ALG) on lymphocyte function in the presence or absence of complement was investigated by comparing (i) ALG-induced blast-cell transformation, (ii) lymphotoxin production and (iii) its effect on PHA-induced blast-cell transformation, and on (iv) lymphotoxin production and (v) its cytolytic effect on lymphocytes. The most striking observation was that even extremely low doses of ALG were able to suppress significantly the lymphotoxin production without affecting other lymphocyte functions or cell viability; this effect was found to be independent of complement. From the comparisons of the mitogen-induced lymphocyte function in the presence of varying doses of ALG and PHA it can be concluded that ALG exerts its inhibitory effect on lymphokine production most likely by interfering with mitogen recognition rather than lymphokine secretion.     

Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor

The effect of mouse epidermal growth factor (mEGF) on the synthesis of glycosaminoglycans and glycoproteins by human fibroblasts has been studied. The addition of physiological concentrations (10^?9 M) of mEGF to quiescent cultures preincubated in the absence of serum was found to elicit an increased incorporation of ³H-glucosamine into the glycosaminoglycans and glycoproteins of both the cellular and extracellular fractions. Although the growth response to the factor, as measured by DNA replication, was minimal under these conditions as compared with the effect of serum, the mEGF-induced incorporation of glucosamine into these cellular constituents and into the extracellular glycoproteins was comparable to that elicited by serum shift-up. Serum, however, caused a significantly larger incorporation of glucoasmine into extracellular, acid-soluble glycosaminoglycans, which were shown to contain hyaluronic acid as the major component. As previously demonstrated, the growth response to mEGF can be enhanced several fold by an mEGF-binding arginine esterase, which is normally associated with the factor in vivo, and by ascorbate. The esterase was found to increase markedly the mEGF-induced incorporation of glucosamine into extracellular hyaluronic acid, while the addition of ascorbic acid did not significantly alter glucosamine incorporation.