Guinea Pig Hepatoma Research Articles

Preparation of Plasma Membranes From Line 10 and Line 1 Guinea Pig Hepatomas

Gentle homogenization followed by differential and density gradient centrifugation was used to purify line 10 and line 1 guinea pig hepatoma plasma membranes in the form of ghosts. Yields of 15--25% allowed enough membranes to be obtained from a single ascites tumor-bearing animal for immunologic and biochemical studies. Although the plasma membrane marker enzyme (Na+ + k+)atpase was present in normal concentrations in both line 10 and line 1 hepatomas, 5'-nucleotidase was reduced over 100-fold in both tumors and phosphodiesterase I was increased 210-fold in the line 10 hepatomas.

Thermographic Tumor Detection Enhancement Using Microwave Heating

Infrared thermography offers a viable alternative to X-ray mammography for early breast cancer detecetion if the inherent Iow sensitivity of the technique can be improved. This paper presents results which indicate that the sensitivity of thermography is increased by irradiating the examined area with microwaves. This arises because of selective absorption characteristics of the particular tumor tissue investigated; specifically, it has been observed that in situ irradiation of transplantable guinea pig hepatoma, using 2450-GHz microwave radiation, results in a tumor temperature rise of 5.5/spl deg/C and a rise of 2.5/spl deg/C in the surrounding healthy tissue. This spatial gradient of 3/spl deg/C compares with the relevant unheated spatial gradient of approximately 0.5/spl deg/C. The microwave-induced increased temperature differential between tumor and healthy tissue is easily observed using a thermovision camera. Data regarding the temporal evolution of spatial temperature distributions associated with tumor tissue before, during, and after microwave irradiation are presented. Additional data are included regarding the heating and cooling rates of microwave-irradiated tumors. The data show conclusively that the specific tumor investigated selectively absorbs microwave energy in situ and exhibits this selective absorption as a thermographically observable increase in local skin surface temperature. The data further show that tumor heating and cooling rates are faster than for healthy tissue.

Effect of protease treatment on the sensitivity of tumor cells to antibody-GPC killing

Treatment of the guinea pig hepatoma designated line-10 with a variety of proteolytic enzymes increased their sensitivity to killing by antibody and guinea pig complement. This increase in sensitivity could not be attributed to the quantity of complement-fixing antibody bound by the cells or to a direct effect on fluid phase complement. Anticomplementary material was removed from the tumor cells by proteolytic enzyme treatment but this material was not solely responsible for the observed increase in sensitivity of the cells to antibody-guinea pig complement killing.

Stimulation of Synthesis and Release of Lipids in Tumor Cells Under Attack by Antibody Plus Complement

Abstract Resistance to antibody-complement- (Ab-C) mediated killing of guinea pig hepatoma cells (line-1 and line-10) correlates with the ability of the cells to synthesize lipids, but not DNA, RNA, protein, or complex carbohydrate. We therefore studied the synthesis and release of macromolecules (especially lipids) by tumor cells under attack by Ab plus C. Ab-sensitized tumor cells (TA) incorporated increased amounts of acetate, glycerol, or fatty acids into complex lipids compared to control cells within 5 to 10 min after the addition of guinea pig C (GPC) to the cells (TAC). TA, TC, or TAΔC (Ab plus heat-inactivated GPC) did not show this effect. No enhancement of DNA, RNA, or protein synthesis in TAC as compared with control cells was noted. Line-1 cells, which are killed by GPC when sensitized with excess anti-Forssman Ab (TAxs), demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to TAxs, before measurable killing of the cells had occurred.

Immunotherapy of a guinea pig hepatoma with vaccines containing mycobacterial fractions

BCG cell walls (CW) were compared to trehalose-6,6′-dimycolate (TDM) in admixture with endotoxin (ET) for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basis, CW were at least as active as ET-TDM. Toxicity of ET and of ET-TDM were greater than that of CW as judged by weight loss after intravenous administration to normal guinea pigs.

Glucan: Attempts To Demonstrate Therapeutic Activity Against Five Syngeneic Tumors in Guinea Pigs and Mice2

Animals with established syngeneic tumor transplants were treated with glucan to study the therapeutic potential of this agent under well-defined experimental conditions. The tumors used were a guinea pig hepatoma, 2 murine fibrosarcomas, a murine melanoma, and a murine adenocarcinoma. All tumors were syngeneic to the host. Living BCG, administered directly into guinea pig tumors, cured all animals, whereas glucan, administered under the same conditions, had no significant antitumor activity. Neither BCG nor glucan, when administered iv, was active against the guinea pig hepatoma. An emulsion prepared with endotoxin, a fraction of mycobacteria related to cord factor, and mineral oil when administered intratumorally was also effective in treatment of line 10 tumor. A similar emulsion, in which glucan was substituted for endotoxin, was inactive, intralesional, ip, or iv administration of glucan was ineffective against the murine tumors. Previous reports of glucan-induced activity against a B16 murine melanoma were not confirmed. BCG was tested against the 2 murine fibrosarcomas and, when given either intratumorally or iv, was found to be effective against one of them.

Identification of Lipids Associated with the Ability of Tumor Cells to Resist Humoral Immune Attack

Certain metabolic inhibitors or chemotherapeutic agents that increase the susceptibility of line-1 or line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of the cells to synthesize lipids. A direct correlation was found between the drug-induced increase in sensitivity to antibody-C mediated killing and the inhibition of the ability of the cells to incorporate acetate, glycerol, and fatty acids into complex cellular lipids. Drug-treated cells recultured in drug-free medium regained their resistance to antibody-C mediated killing; these cells recovered their ability for complex lipid synthesis at this time. Thin layer chromatography of CHCl3:CH3OH lipid extracts from these cells indicated that the drug-induced increase in susceptibility to humoral immune attack correlated with the inhibition of acetate, glycerol, and fatty acid incorporation into cardiolipin and triglyceride in line-10 cells and the inhibition of incorporation of these compounds into cardiolipin alone in line-1 cells. No direct correlation was found between the sensitivity of the cells to humoral immune attack and the ability of the cells to incorporate precursors of lipid synthesis into other lipid moieties (sphyngomyelin, phosphatidyl serine, phosphatidyl choline, phosphatidyl glycerol, or cholesterol esters). The synthesis of cardiolipin and triglycerides, therefore, appears to be associated with the mechanism whereby these tumor cells resist antibody-C mediated killing.

Correlation between the Ability of Tumor Cells to Resist Humoral Immune Attack and Their Ability to Synthesize Lipid

Agents that increase (certain metabolic inhibitors, chemotherapeutic agents, and x-irradiation), decrease (hormones), or have no effect (hyperthermia) on the susceptibility of line-1 and line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of these tumor cells to synthesize macromolecules. A correlation was found between the drug-induced increase in sensitivity of these cells to antibody-C mediated killing and the loss of their ability to incorporate fatty acids into complex cellular lipids. Similarly, the hormone-induced increase in resistance of the cells to killing was accompanied by an enhancement in complex lipid synthesis by these cells was also observed after the cells were exposed to physical means of insult (x-irradiation or hyperthermia). No correlation was found between the sensitivity of the cells to antibody-C mediated killing and their ability to synthesize DNA, RNA, protein, or complex carbohydrate, or their capacity for de novo lipid synthesis as measured by incorporation of acetate and glycerol into cellular macromolecules. The assembly of free fatty acids into complex lipid moieties is therefore proposed to be of fundamental importance for the ability of the tumor cells to resist humoral immune killing.

Increased Susceptibility of Tumor Cells and Chicken Erythrocytes to Lysis by Antibody and Complement after Treatment with Aminoethylisothiouronium Bromide Hydrobromide (AET)

After treatment with aminoethylisothiouronium bromide hydrobromide (AET), the ascites forms of the diethylnitrosamine-induced guinea pig hepatomas, line-1 and line-10, were susceptible or more susceptible to killing in vitro by certain combinations of tumor-specific or IgM anti-Forssman antibody and either human or guinea pig complement. Since AET could be toxic to either cell line, conditions of pH, concentration of AET, and duration of exposure of the cells to the reagent were determined that resulted in enhanced susceptibility without significantly affecting cell viability. Chicken erythrocytes (CE) also were tested and AET-treated cells found to be more susceptible to lysis by IgM anti-Forssman antibody and guinea pig complement. The enhancement apparently was not due to increased ability of AET-treated CE to fix antibody. In contrast to CE, the lytic susceptibility of sheep cells was not affected by AET treatment. In addition to AET, several drugs and enzymes that also can affect the susceptibility of the tumor cells to antibody and complement were tested and found to be ineffective against CE. Since AET reputedly acts directly on the cell surface, it seems reasonable to assume that the increased susceptibility of the tumor cells and CE to antibody and complement may result from a modification of the cell surface.

Immunotherapy of a guinea pig hepatoma with living BCG.A frozen liquid and a lyophilized preparation are equally active

A freeze dried preparation of BCG with a low ratio of living to dead organisms (LV) was compared to a frozen liquid preparation with high viability (HV) for its ability to eradicate established dermal tumors and microscopic lymph node metastases in guinea pigs. The cure rate achieved by the intralesional injection of LV-BCG did not differ significantly from that of HV-bcg when similar numbers of viable organisms were injected.

Metabolic Requirements for Hormone-Induced Resistance to Antibody-Complement Mediated Killing of Tumor Cells

Line-1 guinea pig hepatoma cells are susceptible to killing by anti-Forssman IgM antibody plus guinea pig complement (GPC). When these tumor cells are incubated with insulin, epinephrine, hydrocortisone, or prednisolone, the cells show a marked reduction in their susceptibility to antibody-C-killing. If the ability of the cells to synthesize DNA, RNA, and protein is impaired by pretreatment with metabolic inhibitors, x-irradiation, or culture in nutrient-deficient media, the hormones are no longer effective in rendering the cells resistant to killing. If only DNA synthesis is impaired, but not RNA and protein synthesis, the hormones are effective. The inability of cells inhibited in their macromolecular synthesis to be rendered resistant to killing after hormone treatment is not due to an inability of the cells to bind hormone.

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.

Susceptibility to and escape from complement‐mediated lysis of guinea‐pig hepatoma line‐10

Rabbit xenoantisera produced against diethyl-nitrosamine-induced strain-2 guinea-pig hepatoma line-10 cells (L-10) according to various immunization schedules were compared for their cytotoxicity on L-10 cells in the presence of guinea-pig complement. The highest activity was obtained with antisera (Cx-1) produced by repeated intravenous injections of living L-10 cells at high cell dosage, whereas intramuscular injections of living or glutaraldehyde-treated L-10 cells at similar frequency and cell dosage were less effective for the production of cytotoxic antibodies against L-10 cells. Intravenous injections of smaller cell doses were less effective. The cytotoxic antibody in Cx-1 antiserum was shown to be IgG by various methods including gel filtration on Sephadex G-200, ion exchange chromatography and immunoelectrophoresis of purified tumor-specific antibodies. It was concluded that L-10 cells can be lysed by guinea-pig complement and tumor-specific antibodies (IgG). Some antisera contained IgM antibodies which were not cytotoxic. A decrease in susceptibility of L-10 cells to complement-mediated lysis was observed when the cells were maintained in vivo for a long period (more than 20 passage generations). This was due to a lower density of tumor antigens on the cell surface. When tumor cells were treated with a second antibody directed against rabbit IgG or F(ab')2, cytotoxicity of Cx-1 antisera was completely abolished.

Immunotherapy of a guinea pig hepatoma with ultrasonically prepared mycobacterial vaccines

Oil-in-water emulsions containing either mycobacterial cell walls (CW) or killed whole cells attached to the oil droplets were prepared by ultrasonication and by a modified grinding procedure. The immunotherapeutic potency of the ultrasonically prepared vaccines was at least as great as that of similar vaccines prepared by emulsification in a tissue grinder. Among the advantages of ultrasonication over grinding for the preparation of mycobacterial cell and cell wall vaccines are simplicity and the ease with which sterility may be maintained. Both the ultrasonic and modified grinding methods are less time-consuming than published procedures for preparing mycobacterial vaccines.

Cytotoxicity of guinea-pig lymphoid cells against guinea-pig hepatoma cells in tissue culture

The cytotoxic effect of guinea-pig lymphoid cells on guinea-pig hepatoma cell lines in tissue culture was investigated, using the microplate technique of Takasugi and Klein (1970). The effect of lymphoid cells from guinea-pigs immunized against tumor cells was compared to that of cells from normal controls. Several ratios of effector to target cells (10 : 1, 50 : 1, 150: 1, 250 : 1) were used. In Hartley guinea-pigs immunized with allogeneic tumour cells, peripheral blood lymphoid cells from 14/16 animals showed significant cytotoxicity against that tumour in culture. In a syngeneic tumour/host system, 7/13 animals showed cytotoxicity. Spleen cells gave less consistent results in both systems. The cytotoxic activity of subpopulations of immune lymphocytes against tumour cells in vitro was investigated. It was found that although both T-cell-enriched and T-cell-depleted cell populations exhibited cytotoxicity against tumour cells, the unfractionated cell population was the most effective. This suggests that some degree of cell cooperation may be involved in the cytotoxicity. Antibody-dependent cellular cytotoxicity was also obtained. A T-cell-depleted population of normal cells was shown to be cytotoxic to tumour cells in the presence of serum from immune animals. This type of cytotoxicity could be obtained concomitantly with cell-mediated cytotoxicity in the same animals.

Lysis of Tumor Cells by Antibody and Complement

Abstract The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoisobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complement components. These observations indicate that 86Rb release is not a reliable indicator of cytotoxicity.

Persistence of Immunoglobulin and Complement Components C4 and C3 Bound to Guinea Pig Tumor Cells

Guinea pig hepatoma cells (line-10) growing as ascites were studied for the presence of immunoglobulin, C4, and C3 (components of complement) on their surfaces. Immunoglobulin, C4, and C3 content increased with length of time spent in the peritoneal cavity. The persistence of these factors bound in vivo or in vitro was also determined. Complement-fixing (CF) activity of cellbound antibody disappeared from the surface more rapidly at 37 degrees C than at 4 degrees C; the continued presence of cellbound immunoglobulin in non-complement-fixing form could be demonostrated at either temperature. No CF activity was released into the medium. C4 and C3 were released into the medium and could be demonstrated in the medium by immunochemical methods.

Shared Antigens between Human Malignant Melanoma Cells and Mycobacterium Bovis (BCG)

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.

Regression of Established Tumors and Induction of Tumor Immunity by Intratumor Chemotherapy

The inoculation of a mixture of drugs and guinea pig hepatoma cells (line-10) induced tumor-specific immunity in about 20% of guinea pigs. When guinea pigs with established intradermal tumors were given various drugs ip, no cures were observed; in contrast, multiple intralesional injections of actinomycin D, 1,3-bis(2-chlorethyl)-1-nitrosourea, adriamycin, mitomycin C, and melphalan were effective in curing animals of their intradermal tumors at a time when there were tumor cells in the draining lymph nodes; dimethyl-triazenoimidazole carboxamide, methotrexate, 5-fluorouracil, and 6-mercaptopurine were not effective. More than 80% of the cured animals were immune to rechallenge with 10(6) line-10 tumor cells.

Lysis of Tumor Cells by Antibody and Complement

Abstract The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered them susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could be reversed by incubating the treated cells at 37°C (but not at 0°C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffective in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, β-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase. Enzyme pretreatments that rendered the cells sensitive to killing by antibody and guinea pig complement also enhanced the sensitivity of the cells to the killing action and human complement. In addition the neuraminidase enzyme preparations from Vibrio cholorea or influenza virus increased the sensitivity of the line-10 cells to killing by antibody and human complement, but not to killing by antibody and guinea pig complement.