Correlation between lipid synthesis in tumor cells and their sensitivity to humoral immune attack.

Prolonged incubation of two antigenically distinct, chemically induced guinea pig hepatomas with relatively high concentrations of chemotherapeutic drugs or metabolic inhibitors increases their susceptibility to killing by antibody and complement. This effect is reversible when the cells are cultured in the absence of the drugs. The drug-induced sensitivity and the ability of the cells to recover their resistance to killing are directly correlated to their ability to synthesize complex lipids.

Efficacy of chemotherapeutic treatment of guinea pig hepatoma reflected in host's serum lipid composition.

A correlation was found between the highly polar phospholipid (HPPL) content of serum lipids and the growth and metastasis of the line-10 hepatoma in strain 2 guinea pigs. Animals whose tumors were treated by intralesional injection of decarbazine (DTIC) or saline showed progressive tumor growth and metastasis resulting in death within 120 days of tumor implantation. The percent HPPl in the serum lipids of these animals rose to 40% by 80 days and remained elevated above 20-25% until death. Animals whose tumors were treated by intralesional injection of adriamycin were either cured of their tumors (67%) or showed little or no tumor growth. The percent HPPL in the serum lipids of these animals remained between 3 and 12% at all times. Adriamycin or DTIC injected into non-tumor-bearing animals resulted in an HPPL content of serum lipids that was not significantly different from that of control, saline-inoculated animals.

Plasma membrane and intracellular lipid synthesis in tumor cells rendered resistant to humoral immune killing after treatment with hormones.

Line-10 hepatoma cells from Sewall Wright guinea pigs are sensitive to killing by antibody plus human complement. Hormones that decrease the sensitivity of the cells to antibody-complement-mediated killing (insulin and hydrocortisone) were examined for their effects on the ability of the cells to synthesize and incorporate specific lipids into plasma membrane and intracellular membrane fractions. Cells that had been rendered resistant to antibody-complement-mediated killing following incubation for 1 hour with either of the hormones were enhanced in their incorporation of newly synthesized L-alpha-phosphatidyl serine, L-alpha-phosphatidyl choline, and triglycerides into the plasma membrane as well as L-alpha-phosphatidyl choline, L-alpha-phosphatidyl serine, and cholesteryl ester into mitochondria, endoplasmic reticulum, nuclear membrane, or microsomes; these cells were inhibited in cardiolipin synthesis. Cells cultured for 4 hours with hormone regained their sensitivity to antibody-complement-mediated killing and reverted to control levels in their ability to synthesize and incorporate lipids into plasma and intracellular membranes. These data suggest that agents that increase the resistance of the tumor cells to humoral immune killing stimulate the synthesis and incorporation of specific complex lipids into plasma membrane and intracellular organelles; these effects are generally opposite those observed after treatment with agents that increase the sensitivity of the cells to antibody-complement-mediated killing (metabolic inhibitors).

Plasma membrane and intracellular lipid synthesis in tumor cells rendered sensitive to humoral immune killing after treatment with metabolic inhibitors.

Line 10 guinea pigs hepatoma cells are resistant to killing by antibody and guinea pig complement. Metabolic inhibitors (adriamycin and actinomycin D) that increase the sensitivity of the cells to antibody-complement (C) killing were examined for their effects on the ability of the cells to synthesize and incorporate specific lipids into plasma membrane and intracellular membrane fractions. Drug-treated cells that had been rendered sensitive to antibody-C killing were inhibited in their incorporation of newly synthesized phosphatidylcholine and cholesteryl ester into the plasma membrane, as well as incorporation of phosphatidylcholine, cardiolipin, cholesteryl ester, and triglyceride into certain intracellular organelles including mitochondria, endoplasmic reticulum, nuclear membrane, or microsomes. Drug-treated cells recultured in the absence of the drug regained their ability to resist antibody-C killing and to synthesize and incorporate lipids into plasma and intracellular membranes. These data suggested that agents modifying the sensitivity of the tumor cells to humoral immune killing have a concomitant effect on plasma membrane and intracellular lipid synthesis.

Correlation between the ability of tumor cells to incorporate specific fatty acids and their sensitivity to killing by a specific antibody plus guinea pig complement.

Line-1 diethylnitrosamine-induced guinea pig tumor cells can be rendered sensitive to killing by rabbit anti-Forssman IgM antibody plus guinea pig complement (GPC) or antitumor antibody plus GPC following prolonged incubation (17 hr ) of the cells with one of several metabolic inhibitors. Compared to control cells, these cells have been shown to be inhibited in their ability to incorporate fatty acids into complex cellular lipids, which suggested that lipid synthesis is of fundamental importance for the ability of the tumor cells to resist humoral immune killing. In this study, drug-treated cells that were rendered sensitive to killing by anti-Forssman antibody plus GPC, but not antitumor antibody plus GPC, were inhibited in their incorporation of saturated (palmitic or stearic acid), but not an unsaturated, fatty acid (linoleic acid). These data suggested that the fatty acid composition of specific lipids may also be important for the resistance of these tumor cells to killing by antibody and complement.

Physical and chemical composition of subcellular fractions from tumor cells treated with metabolic inhibitors or hormones.

Line 10 guinea pig hepatoma cells are resistant to killing by antibody plus guinea pig complement but not by antibody plus human complement. Agents that increase (metabolic inhibitors) or decrease (hormones) the sensitivity of the cells to killing by antibody plus complement were examined for their effects on the chemical, physical, and enzymic composition of the cells. The effects of these agents on the chemical and enzymic characteristics of isolated plasma membrane and intracellular fractions of these cells were also measured. Adriamycin treatment resulted in a decrease in the amount of ribonucleoprotein and smooth endoplasmic reticulum isolated from the cells as compared to untreated cells. Hormone (insulin or hydrocortisone)-treated cells were enhanced in their yield of this fraction but were decreased in their yield of mitochondria as compared to controls. Generally, the drug-treated cells were decreased, whereas hormone-treated cells were enhanced, in protein, lipid phosphate, and total phosphate content, as compared to untreated controls. This pattern was also noted in the plasma membrane fraction of the cells and, in several cases, in intracellular membrane fractions. These data suggest that the protein, lipid phosphate, and total phosphate content of the plasma membrane and intracellular membranes of these cells may correlate with their ability to resist humoral immune attack.

Inhibition of antibody-complement-mediated killing of tumor cells by hormones.

Line 1, a chemically induced guinea pig hepatoma, is susceptible to killing by anti-Forssman immunoglobulin M antibody and guinea pig complement. When these tumor cells are pretreated at 37 degrees with 10(-4) to 10(-11) M concentrations of the polypeptide hormone insulin, with the catecholamine L-epinephrine-HCl, or with the glucocorticoid steroids hydrocortisone sodium succinate or prednisolone sodium succinate, the cells show a marked reduction in their suseptibility to killing by antibody and guinea pig complement; pretreatment at 0 degrees is ineffective. Similar results were obtained with another antigenically distinct guinea pig hepatoma (line 10) when tested with anti-Forssman immuno-globulin M or specific antitumor antibodies and human complement. The ability of the hormones to render the cells resistant is dependent on time, temperature, and hormone concentration. The effect of hormone treatment is maximal between 30 and 60 min and is reversible within 4 hr even in the continued presence of hormone. Treatment of line 1 cells with up to 10,000-fold greater concentrations of the less biologically active or inactive analogs, DL-epinephrine, beta-estradiol, testosterone, or proinsulin has no effect on the susceptibility of the cells to killing by antibody and guinea pig complement. The effect of hormone treatment is not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.

PIP: Various aspects of hormone treatment of tumor cells are reported; it is shown that following treatment with certain hormones, the cells are less susceptible to killing by antibody and complement. The diethylnitrosamine-induced guinea pig hepatoma, designated Line 1, is susceptible to killing by anti-Forssman immunoglobulin M (IgM) antibody and guinea pig complement (GPC) but not by specific antitumor antibody and GPC. The antigenetically distinct Line 10 hepatoma, when sensitized with either antibody, is susceptible to killing by human complement (HUC) but not by GPC. Strain 2 of Servall-Wright male guinea pigs were used. 2 antigenetically distinct diethylnitrosamine-induced hepatic tumors (ascites form), Lines 1 and 10, passed in Strain 2 guinea pigs, were collected and suspended in RPMI 1640-20% FCS. Toxicity assays were performed in VBS-gel. The hormones used were hydrocortisone sodium succinate, prednisolone sodium succinate, NSC9151, bovine insulin, L-epinephrine methyl ether HC1, DL-epinephrine, beta-estradiol, testosterone, pork insulin, chicken insulin, pork proinsulin, pork DAA insulin, and the A and B chains of pork insulin. Tumor cells were cultured in 10-ml volumes of RPMI 1640-20% FCS in plastic Petri dishes. After incubation, cell cultures were washed 5 times in VBS-gel and tested for their susceptibility to killing by antibody and complement. Rabbit antiserum to sheep Forssman antigen was prepared and stored at -20 degrees until used. Tumor specific rabbit Antilines 1 and 10 antisera were prepared and similarly stored. Results of tests show that Line 1 tumor cells incubated in a medium containing the polypeptide hormone, insulin, the catecholamine, L-epinephrine HCl, or the glucocorticoid steroids, hydrocortisone sodium succinate, or prednisolone sodium succinate were rendered resistant to killing byanti-Forssman IgM antibody and GPC. This effect was dependent on hormone concentration, temperature, and time. Effects were reversible. Similar results were obtianed with Line 10 cells under attack by specific antitumor and HUC or anti-Forssman antibodies. Less physiologically active analogs of the hormones did not have this effect. Tumor cells showed maximum resistance within 30-60 minutes of exposure to the hormones and reverted to the sensitive state within 4 hours. Resistance of the cells to killing was observed at 37 degrees but not at 0 degrees. It is concluded that the effect of hormone treatment was not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.

Complete and apparently specif local tumor regression using syngeneic or xenogeneic "tumor-immune" RNA extracts.

Syngeneic and xenogeneic RNA-rich extracts of lymphoid tissues were used in an immunotherapeutic regimen to treat strain 2 guinea pigs that were given intradermal injections of a uniformly lethal dose (1 x 10(6)) of line 10 diethylnitrosamine-induced transplantable hepatoma cells. When 1 X 10(7) syngeneic nonsensitive peritoneal exudate cells, 2.5 mg RNA from line 10-immune strain 2 guinea pigs or line 10-immune Rhesus monkeys, and 1.0 mg of a line-10 tumor-specific antigen preparation were injected s.c. under the tumor cells injected 5 days previously, complete local tumor regression in all treated animals was observed. If either nonsensitive peritoneal exudate cells, RNA, or line 10 tumor-specific antigen was omitted, or if Escherichia coli RNA or RNA from animals sensitized to a different tumor (line 1) was used, little or no tumor regression was observed, suggesting that the action of the RNA may have resulted in an antitumor response specific for the noplasm being treated. The long-term tumor-free survival of all treated animals indicates that the action of the RNA is systemic, since metastases are known to occur frequently by the time our therapeutic regimen was given. Also, in testing the biological activity of the "tumor-immune" RNA in the in vitro cell-migration-inhibition assay, both the syngeneic and xenogeneic RNA extracts could transfer tumor-specific immunological sensitivity, as demonstrated by the elaboration of migration-inhibitory factor by the RNA-treated nonsensitive peritonial exudate cells in the presence of the line 10 tumor-specific antigen.

Kinetics of hormone-induced tumor cell resistance to killing by antibody and complement.

Line 1, a chemically induced guinea pig hepatoma, is susceptible to killing by anti-Forssman immunoglobulin M antibody and guinea pig complement. When these tumor cells are pretreated with insulin, L-epinephrine, hydrocortisone, or prednisolone, the cells show a marked reduction in their susceptibility to antibody-complement-mediated killing within 15 to 60 min; this effect reverses within 4 hr in the continued presence of hormone. Maximal binding of the hormones to the line 1 cells was observed within 60 min. However, the hormones remained bound to the cells after 4 hr of incubation, suggesting that line 1 cells incubated in the continued presence of hormone revert to the susceptible state despite the persistence of cell-bound hormone. Hormone-treated tumor cells, washed free of hormone and reincubated in hormone-free medium, lost nearly all their bound hormone within 15 to 30 min of washing. These cells, however, remained resistant to antibody-complement-mediated killing for up to 2 hr after washing. Line 1 cells, reverted in the continued presence of hormone, remained susceptible to killing by antibody and guinea pig complement after reexposure to the same, but not to a different, hormone. Hormone-treated cells reverted after prolonged incubation in hormone-free media; however, they were rendered resistant to killing after reexposure to the same hormone. The temporary refractoriness of reverted cells to further hormone stimulation was not due to an inability of the cells to bind hormone.

Cell growth-dependent variation in the sensitivity of human and mouse tumor cells to complement-mediated killing.

Cells removed from asynchronous cultures during lag, log, and stationary phases of growth were found to differ in their sensitivity to antibody/complement-mediated killing. The human lymphoblastoid line, Raji, was relatively more susceptible to killing by human anti-HLA antibody plus rabbit complement during the lag and log phases of growth, while the human lymphoid cell line, PY, was relatively more susceptible to rabbit antilymphocyte serum or human anti-HLA plus rabbit complement during the log and late-log phases of growth. The mouse mastocytoma cell line, P815, was relatively resistant to killing by rabbit anti-P815 plus guinea pig complement during the middle log phase of growth. The variation in sensitivity of the three cell lines was dependent upon the concentration of antibody used to sensitize the cells but not due to differences in antigen expression, antigen density, or net synthesis of DNA, RNA, protein, complex carbohydrate, or lipid-containing macromolecules. These data suggest that the variability in susceptibility of the cells for complement-mediated killing is due to changes in physiological and/or physicochemical properties of the cells which either affect the ability of the cells to repair complement-mediated damage or nullify the activity of cell-bound complement.

Effect of inhibiting DNA, RNA, and protein synthesis of tumor cells on their susceptibility to killing by antibody and complement.

A number of metabolic inhibitors and chemotherapeutic agents have been found to increase the sensitivity of a chemically induced guinea pig hepatoma (line 1) to killing by antibody and complement. We have investigated whether the mechanism whereby these drugs increase sensitivity to killing is attributable to their primary action of inhibiting DNA, RNA, or protein synthesis. Line 1 cells incubated for 1, 4, or 17 hr with actinomycin D (25 microng/ml), adriamycin (40 microng/ml), or puromycin (5 micron/ml) or with 5-fold lower concentrations of these drugs were maximally inhibited (greater than 90%) in their ability to synthesize DNA, RNA, and protein within 1 hr. However, only cells incubated for 17 hr with the high concentrations of drugs showed increased sensitivity to killing by antibody and complement. Line 1 cells incubated with high concentrations of these drugs of 17 hr, washed, and resuspended in drug-free medium recovered their resistance to killing by antibody and complement within 4 hr. These cells ever after culture for 24 hr in drug-free medium did not regain their ability to synthesize DNA, RNA, or protein. A similar lack of correlation between synthesis of these macromolecules and sensitivity to antibody-complement-mediated killing was observed after the cells were treated with physical agents that inhibit macromolecular synthesis. Both heat-treated and X-irradiated cells were inhibited in their ability to synthesize DNA, RNA, and protein immediately after treatment; however, only X-irradiated cells (6 and 16 hr postirradiation) were increased in their sensitivity to antibody-complement-mediated killing. Our data show that the ability of line 1 tumor cells to resist humoral immune attack does not depend solely on their ability to synthesize DNA, RNA, or protein.

Humoral immune killing of nucleated cells: mechanisms of complement-mediated attack and target cell defense.

The killing or lysis of nucleated cells, erythrocytes, bacteria, and other targets (e.g., liposomes) by antibody and complement is the result of complex series of actions and interactions between antibody, components of the complement system, and the cell. The complement attack mechanism has strict qualitative and quantitative requirements. For efficient activity, sufficient amounts of antibody must bind to the cell, the antibody must be of the complement-fixing type, and sufficient amounts of complement components must be activated and fixed to the surface of the target cells. Nucleated cells of different types differ in their sensitivity to the cytotoxic action of complement. This difference in sensitivity may be attributed to differences in metabolic properties and/or the chemical and physical composition of the cells. Since complement action occurs primarily on or in the cell membrane, the properties of the cell which may affect the outcome of complement-mediated attack should be linked to cell membrane function and integrity. The relationship between the susceptibility of nucleated cells to complement-mediated killing and the chemical and metabolic properties of the cells will be discussed in this review.

Role of membrane lipids in the immunological killing of tumor cells: I. Target cell lipids.

The metabolic and physical properties of tumor cells that are associated with their ability to resist or escape from immune attack have been investigated. The susceptibility of P815 murine mastocytoma cells to immune killing can be modulated. Culturing the cells with adriamycin or with hydrocortisone increases or decreases, respectively, the sensitivity of the cells to killing by antibody (Ab) plus complement (C); in addition, culturing the cells with mitomycin C or hydrocortisone increases or decreases, respectively, the sensitivity of the cells to killing by cytotoxic T lymphocytes (CTL). The susceptibility of the cells to Ab-C killing correlates with the ability of the cells to synthesize complex cellular lipids, but not DNA, RNA, protein, or carbohydrate. Further, tumor cells rendered sensitive to Ab-C killing by adriamycin are decreased in total lipid content and in their cholesterol/phospholipid mole ratio; hydrocortisone-treated resistant cells showed the opposite effects. The ability of tumor cells to resist CTL killing did not correlate with their total cellular lipid synthesis, but did correlate with the synthesis and composition of specific cellular phospholipids. In addition, tumor cells increased in sensitivity to Ab-C killing exhibited an increase in cell surface membrane fluidity, whereas cells increased in susceptibility to CTL attack showed an increase in their net negative cell surface charge density. These data show certain unique chemical and physical properties of tumor cells to be of fundamental importance for their ability to resist either humoral or cell-mediated immunologic attack; modulation of one or another of these cellular properties results in a change in the cells' susceptibility to immune killing by antibody plus C or by cytotoxic T lymphocytes.

Role of membrane lipids in the immunological killing of tumor cells: II. Effector cell lipids.

Peritoneal macrophages (M phi) from mice become cytotoxic after incubation in lymphokine (LK)-rich supernatants of antigen-stimulated spleen cell cultures. Tumoricidal activity is evident with M phi treated with LK for 4 hr, becomes maximal after 8-12 hr incubation and decreases to control levels by 24-36 hr. To gain insight into LK-induced functional changes, the lipid composition of M phi cultured with LK for 0-36 hr was analyzed by high pressure liquid chromatography. LK induced marked changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids increased 2- to 3-fold after 8 hr when the cells showed maximal tumoricidal activity. Cellular lipid and fatty acid content returned to control levels by 24 hr when the M phi had lost tumoricidal activity. These changes were not observed with equal numbers of M phi cultured in control supernatants. To analyze further the role of CHOL and unsaturated fatty acids in M phi tumor cytotoxicity, M phi were enriched in CHOL or linolenic acid (18:3) and tested for their ability to kill 1023 tumor cells. Within 1 hr of culture, M phi showed a 3- to 4-fold increase in CHOL or 18:3 content. 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acids were cytotoxic. CHOL-enriched M phi were not tumoricidal; indeed, these cells were inhibited in their killing after treatment with LK compared to M phi cultured in delipidized medium with LK alone. These results suggest that UFA aids, whereas CHOL negates, expression of M phi tumor cytotoxicity.