Monoclonal antibody against tumour-derived cell line shows heterogeneity of altered hepatocytes during afb1-induced carcinogenesis

Monoclonal antibodies have been raised against a tumour cell line (JBI) derived from an aflatoxin B₁-fed rat liver. One antibody MRC TU/Jl has been characterized in terms of its immunohistochemical staining in sections of control and AFB,-fed liver and by the proteins which it immunoprecipitated. The antibody reacted with bile duct epithelium in all sections. In control liver sections there was staining of hepatocyte membranes, with increased stain in periportal regions in animals ?12 weeks old. In AFB,-fed liver sections, areas of biliary hyperplasia were stained and after 4 weeks on AFB₁-diet, foci of altered hepatocytes were visible. These were negative, positive or heterogeneous with respect to antibody stain. The antibody reacted much more strongly with adenomatous and cholangiomatous tissue compared with trabecular regions of hepatic tumours which developed in animals after 14 weeks on AFB, diet and 9 months on control diet. After immunoprecipitation of ¹⁴C-leucine-labelled proteins from a JBI cell lysate by MRC TU/Jl, 5 bands in the molecular weight range 43,000-49,000 daltons were visualized on 10% SDS-PAGE gels. A similar result was obtained with ¹⁴C-glucosamine in place of leucine, indicating that all 5 bands were glycoproteins. Deglycosylation of JBI cell lysates with CNBH₁-/IO₄- prevented immunoprecipitation of all bands, suggesting that MRC TU/JI recognized antigenic sites on the carbohydrate portions of the molecules.     

Identification of glycoproteins expressing tumour-associated PNA-binding sites in colorectal carcinomas by SDS-GEL electrophoresis and PNA-labelling

Many tumour-specific antigens in gastrointestinal cancers have carbohydrate immuno-determinants. These epitopes can be identified by lectins and monoclonal antibodies. By using fluorescein-isothiocyanate (FITC)-conjugated peanut agglutinin (PNA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we have investigated glycoproteins carrying altered carbohydrate epitopes in normal and carcinomatous human colorectal mucosa. In normal mucosa PNA stained goblet cell glycoconjugates in the supranuclear (Golgi) distribution. After neuraminidase pretreatment PNA stained actual mucin goblet itself at all levels of the crypts. Colorectal carcinomas displayed a strong and direct binding of PNA to apical cell membranes of carcinomatous cells and intraluminal secretions. Analysis of the glycoproteins by SDS-PAGE and PNA-labelling revealed four carcinoma-associated glycoproteins (26kD, 32kD, 35kD and 50kD). In addition, four glycoproteins (29kD, 30kD, 33kD and 36kD) common to normal and carcinomatous colorectal mucosa could be identified. All of these glycoproteins differed in their molecular weight from those in red cell controls which bind PNA only after desialylation. The study shows that the expression of PNA-binding sites in colorectal carcinomas signifies a cancer-associated carbohydrate alteration. Four carcinoma-associated glycoprotein antigens could be detected by this lectin. The antigens we have identified might be useful in the isolation and purification of more selective reagents for the serologic detection of colorectal cancer.     

Alterations in glycoproteins and lipids in azaserine-induced acinar cell carcinoma of rat pancreas

Glycoproteins and lipids of rat pancreatic acinar cell carcinomas maintained in nude mice and in cell culture, were analyzed. The tumor contained significantly elevated levels of glycoproteins when compared with their normal counterparts. SDS-PAGE of tumor glycoproteins revealed that there were increased amounts of small molecular weight glycoproteins and the tumor also contained a 51,000 dalton glycoprotein which was not detected in the pancreas, liver or the sera of the control animals. The tumor in nude mice and cancer cells in culture had decreased lecithins and triglycerides, and increased amounts of free fatty acids, and both free and esterified cholesterols. The results indicate that altered glycoprotein and lipid compositions represent some of the characteristic features of the acinar cell carcinoma.     

Autonomous cholesterol biosynthesis in murine hepatoma. A receptor defect with normal coated pits

These studies indicate that autonomous cholesterol biosynthesis by hepatocellular carcinoma may result from absent or defective receptors for chylomicron remnants on the surface of the malignant hepatocytes. In vivo, DAB2 hepatoma or liver were perfused with chylomicron remnants labeled with tritiated palmitic acid. Normal liver had chylomicron remnant uptake/gm tissue that was ten times that of hepatoma. In vitro studies using isolated hepatocytes and cultured DAB2 hepatoma cells showed similar results. Uptake of chylomicron remnants labeled with 3H-palmitic acid by normal hepatocytes during a 4-hour period was ten times that of hepatoma cells. Both in vivo and in vitro differences were statistically highly significant (P less than 0.005). Since many surface receptors are related to the coated pits, the cellular membranes of both neoplastic and normal liver cells were examined by electron microscopy. Coated pits were present in both the hepatoma and normal liver cells and occupied 2.61% and 2.65% of the cell surface, respectively. The defective uptake of chylomicron remnants by DAB2 hepatoma appears to be related to the chylomicron remnant receptor and not to the coated pit-internalization mechanism.     

Pharmacokinetics of the recombinant fusion protein DAB486IL-2 in animal models

The kinetics of the in vitro cytotoxicity of DAB486IL-2, a genetically engineered fusion protein containing a portion of diphtheria toxin and human interleukin-2, were examined in the C91/PL cell line, which constitutively expresses IL-2 receptors. Maximal inhibition of protein synthesis was observed by 4-6 h after DAB486IL-2 addition at a concentration of 300 ng/ml. The tissue distribution, urinary excretion, and plasma pharmacokinetics of DAB486IL-2 in the rat and its plasma pharmacokinetics in the monkey were also examined. In rats the primary site of distribution of [35S]-DAB486IL-2 outside the vasculature appears to be the liver, followed by the kidney, spleen, and lung. Persistence of radioactive material in the liver and urinary excretion of metabolic degradation products suggest that labeled protein is metabolized by hepatic tissue. Following i.v. bolus administration of DAB486IL-2, the initial serum half-life for both the rat and the monkey was approximately 5 min. The overall clearance rate of drug for the two species differed, with DAB486IL-2 being cleared from circulation 2-3 times more rapidly in the monkey. Presence of high levels of neutralizing antibodies to diphtheria toxin in the rat significantly influenced the clearance of bioactive DAB486IL-2. However, the question as to whether the presence of in vitro biological activity for the molecule is masked by the presence of antibodies cannot be clearly answered.     

A 72 kD trophoblast glycoprotein defined by a monoclonal antibody

A novel trophoblast cell surface antigen has been defined by a monoclonal antibody 5T4, raised following immunisation with wheat germ agglutinin (WGA) purified glycoproteins from deoxycholate (DOC) solubilised human syncytiotrophoblast plasma membrane (StMPM). The distribution of the antigen was determined by indirect immunoperoxidase staining of sections of normal organ and placental tissues as well as immunofluorescence and radiobinding assays with a wide variety of cell lines representing differing normal and tumour cell types. In frozen sections of normal full term placenta, 5T4 is strongly expressed only by the syncytiotrophoblast, some extravillous cytotrophoblast and the amniotic epithelium. The 5T4 antigen is apparently not expressed by any maternal component of the placenta nor is it detected in adult liver, lung, bronchus, heart, testis, ovary, brain, or muscle. The antigen is apparently expressed by several specialised epithelia. Immunoprecipitation of radiolabelled StMPM indicated that 5T4 molecules are glycoproteins of mol. wt of approximately 72 kD on SDS-PAGE. 5T4 antigen is selectively expressed by diverse tumour cell lines, including those of developmental origin. The molecular characteristics, relatively restricted normal tissue distribution and expression by certain tumour cell types make this antigen worthy of future study for use as a diagnostic marker of malignancy.     

DAB-induced changes in NMR relaxation times, water and iron content of rat tissue.

T1 relaxation values of rat liver and spleen tissue have been measured over a 6-month period whilst feeding with p-dimethyl-aminoazobenzene (DAB). Measurements of tissue water and iron content have also been made. A small rise in liver T1 value during the early stages of DAB feeding, from a control value of 296 +/- 12 ms to 318 +/- 12 ms after 3 weeks, probably reflected toxic reaction to the diet rather than preneoplastic changes. Spleen T1 value showed a considerable decrease over this same period, from a mean control value of 505 +/- 14 ms to 394 +/- 21 ms after 3 weeks on the diet. The possible origins of this change are discussed.     

Binding of metabolites of dietary 4-dimethyl-aminoazobenzene and 2-methyl-4-dimethylamino-azobenzene to rat liver DNA and protein of subcellular fractions

The binding of metabolites of two related azo dyes of different carcinogenic potency, the carcinogenic 4-dimethylaminoazobenzene (DAB) and the weakly carcinogenic 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB), to rat liver DNA and to subcellular fraction protein was studied following chronic oral administration for 1 to 3 weeks. Different techniques for measuring the amount of DAB metabolites bound to protein were first compared, then the whole study was performed with labelled DAB and 2-Me-DAB (aniline ring-¹⁴C) of moderate specific activity. DAB metabolites were bound to liver DNA to a higher extent than those of 2-Me-DAB. In contrast, the binding of 2-Me-DAB metabolites was equal to or higher than that of DAB metabolites to protein. The amount of protein-bound metabolites was studied on the nucleo-mito-chondrial fraction, microsomes, supernatant, nuclei, chromatin, nucleoplasm, nucleolar fraction and nuclear membrane. Following the administration of both dye diets, the supernatant protein bound the highest level of metabolites. The time-course of binding of DAB metabolites to DNA and protein was different from that of 2-Me-DAB metabolites. These results show the possible involvement of carcinogen-DNA binding in the mechanism of carcinogenesis.     

Sequential effects of chemically different carcinogens,dimethylnitrosamine and 4-dimethylaminoazobenezene,on hepatocarcinogenesis in rats

Induction of liver tumors was studied in rats given carcinogens as follows: (1) N-nitrosodimethylamine (DMN) alone; (2) 4-dimethylaminoazobenzene (DAB) alone; (3) DMN followed by DAB; and (4) DAB followed by DMN. The main findings were: (1) DAB feeding for 0.62 to 2.5 months, followed by DMN feeding for 5 months, induced liver carcinomas more frequently than did DMN alone given for 5 to 10 months, or DAB alone given for 2.5 months. (2) Likewise, DMN feeding for 0.62 or 1.25 months, followed by DAB feeding for 5 months, induced liver carcinomas more frequently than did DMN alone given for 5 to 10 months or DAB alone given for 2.5 months. Interestingly, when DMN was given for a longer time (2.5 months), followed by DAB for 5 months, 50% of the induced liver tumors were non-epithelial. (3) Liver tumors were induced by sequential administration of the two carcinogens in doses that did not induce tumors when each carcinogen was given alone over a comparable time period. (4) The latent period for liver tumor development was shorter in groups that received the two carcinogens sequentially than in those that received DAB or DMN alone for comparable time periods.     

Azoreductase activity of Sprague Dawley and Wistar-derived rats towards both carcinogenic and non-carcinogenic analogues of 4-dimethylaminophenylazobenzene (DAB)

Azoreductase activity towards the hepatocarcinogen p-dimethylaminophenylazobenzene(DAB) and four analogues has been measured in vitro in the liverand caecum of Sprague Dawley (Alpk/SD) and Alderley Park (Alpk/APWistar-derived) rats. Two carcinogenic DAB analogues, 3'-methyl-p-dimethylaminophenylazobenzene(3M) and 6-p-dimethyl-aminophenylazobenzothiazole (6BT) andtwo non-carcinogenic analogues, 4-N-pyrrolidinylazobenzene (4N)and 5-p-dimethylaminophenylazoindazole (5I) have been examined.The azoreductase activity towards DAB of a 9000 g supernatantof liver homogenate was greater in the SD than the AP strainbetween 6 and 13 weeks of age, but comparable to that of APrats at 4 weeks of age. The activity towards DAB fell in bothstrains with increasing age. Animals of both strains fed a riboflavin-lowdiet (2–3 mg kg^–1) had reduced azoreductase activitywith DAB when compared to a standard diet at all ages studied,although the difference was less marked in the AP rats. 3M and4N were azoreduced by the livers of both strains of rat feda standard diet at a rate of 50% of that of DAB, whereas 51and 6BT were cleaved at a much lower rate (5–20%). Allthe chemicals were reduced by an oxygen-insensitive enzyme inthe liver preparation, as has previously been reported for DAB.DAB, 3M and 6BT were reduced at a similar rate to each otherby a fraction containing caecal contents, both in and betweenthe two strains of rat. Similarly, 4N and 51 were reduced bya caecal preparation at a similar rate to each other in andbetween both strains of rat, but at a rate of only 30–50%that shown by DAB, 3M and 6BT. In contrast to the conditionsrequired by the liver azoreductase enzyme, anaerobic conditionswere required for maximal activity of the caecal preparation.Liver azoreductase activity towards all the DAB analogues wasreduced in both strains of rat maintained on a riboflavin-lowdiet, while the caecal azoreductase activity was unaffected.Neither the activity profile observed in vitro in the livernor the caecum was found to correlate with the relative carcino-genicityreported for these compounds, suggesting that other factorsare more important in determining this toxicity for the seriesof azo chemicals examined in this study.     

Saccharide alterations in rat kidney associated with malignant transformation by injection of dimethylnitrosamine.

Renal tumours were induced in dietary-primed rats by injection of dimethylnitrosamine. Control and tumour tissue was excised at varying periods and maintained in short-term organ culture in the presence of 3H- or 14C-fucose. The plasma membranes were then isolated, and the isotopic profiles of normal kidney and renal tumour membrane proteins were established, using polyacrylamide-gel electrophoresis in dodecyl sulphate. Several fucose-containing glycoproteins of the plasma membranes were found to alter upon neoplastic transformation: 4 increased and 3 decreased. The probable identity of 2 of these proteins is indicated: alpha-foetoprotein is one of the glycoproteins which increased, whereas neutral endopeptidase decreased in the tumour membranes. Fluorescein-labelled lectin binding by the kidney tissue was also found to alter upon transformation. The most marked changes were an increase in sialic acid (neuraminidase-sensitive) and galactosamine (Ricinus communis agglutinin Type I) in the nuclei of some neoplastic cells and some hyperplastic-tubule cells.     

Azo-dye binding to proteins and detoxication of p. dimethylaminoazobenzene in mouse and hamster liver

We studied azodye binding to proteins, and at the same time azoreductase and NADPH-cytochrome c reductase activities in the liver of IC and C3H mice and hamsters fed DAB over a long period. Sensitivity of C3H mice to the toxic effects of DAB and resistance of IC mice and hamsters were confirmed by histological examinations. After 2–3 weeks of DAB feeding, the amount of protein-bound azo-dyes was three times higher in C3H mice than in IC mice. For hamsters, limited azo-dye binding to proteins increased gradually up to 6 weeks, then remained nearly constant. In control animals azoreductase activity was five times higher in IC mice than in C3H mice; activity of hamsters was intermediate. NADPH-cytochrome c reductase activity was nearly the same in all three groups, and was in no way related to azoreductase activity. The sensitivity of C3H mice to the toxic effects of DAB could be explained by their weak azoreductase activity and high level of azodye binding to proteins. On the contrary, high azoreductase activity in IC mice would allow detoxication of DAB and prevent or delay its toxic effects. Resistance of hamsters could be linked to the small amount of protein-bound azo-dye; yet, it cannot be explained by their weak azoreductase activity and probably depends on other metabolic processes, which have been considered.     

Hyposialylation of high-molecular-weight membrane glycoproteins parallels the loss of metastatic potential in wheat-germ agglutinin-resistant Friend leukemia cells

From the highly metastatic in vivo-passaged Friend leukemia cells (FLC), WGA-resistant (WR) tumor cell variants were selected. These WR FLC had lost their capacity to metastasize when injected i.v. or s.c. into DBA/2 mice. We have characterized the plasma membrane glycoproteins of the different FLC types by: (i) metabolic labelling with (3H)-galactose; (ii) surface labelling with galactose oxidase-borohydride; (iii) direct binding of (125I)-lectins on glycoproteins separated by SDS-PAGE. The ensemble of these approaches showed that the 100- to 200-kDa glycoproteins of in vivo-passaged FLC and WR FLC exhibited a very similar distribution of the terminal galactose in their oligosaccharide moieties. In contrast, the expression of terminal sialic acid was reduced in WR FLC with respect to in vivo-passaged counterparts as appreciated by: (i) binding experiments with (125I)-WGA; (ii) cathodic shift of the 100- to 200-kDa glycoproteins in 2-dimensional electrophoresis studies, and (iii) thiobarbituric acid assay after FLC treatment with neuraminidase. Moreover, binding experiments with (125I)-LPHA, (125I)-ConA and (125I)-WGA (after Smith degradation) indicated that, in the 100- to 200-kDa region, virtually identical asparagine-linked tri- or tetra-antennary complex-type oligosaccharides were expressed in both cell types. We conclude that the sialylation of high-molecular-weight surface glycoproteins (particularly in the 150-kDa region) is strongly associated with the metastatic potential of FLC, especially to the liver.     

Characterization of plasma membrane shedding from murine melanoma cells

Tumor cells release intact portions of their plasma membranes in the process of membrane fragment shedding. This released material has been shown to inhibit various synthetic functions of normal cells, which may play an important role in certain patho-physiological events occurring in advanced-stage cancer patients. Our studies on metastatic variants of the murine B16 melanoma, B16-F1 (low incidence of lung colonization) and B16-F10 (high incidence of lung colonization) indicate that the shed membrane fragment material is composed predominantly of vesicles, ranging in size from 20 to 100 nm in diameter. The release of membrane fragments represents a small percentage (approximately 16%) of the total shedding of plasma membrane components. Membrane fragments were shed at a higher rate from the highly "metastatic" (colonizing) B16-F10 cells than from poorly metastatic B16-F1 cells, resulting in a 2-fold greater accumulation of membrane fragment material by cultures of B16-F10 cells than by B16-F1 cultures during the 48-hr assay period. The study of various intracellu ar metabolic processes (protein and RNA synthesis, glycosylation, and generation of ATP) required for the shedding of membrane fragments indicated that the shedding event is only dependent on energy when inhibitors of the above processes are present for 2 hr. Treatment of cells with these inhibitors for 8 hr results in cessation of the shedding process, indicating both a limited pool of components to be shed and the requirement for further synthesis of the shed material. Glycoprotein components of the shed membrane fragments were analyzed by SDS-polyacrylamide gel electrophoresis. In addition to quantitative differences, 2 additional bands were present in fluorographs from SDS-PAGE gels from the B16-F10 membrane fragment material which were not present in fluorographs from B16-F1 fragments. The glycoprotein components of shed membrane fragments were shown to represent selected domains of the cell's plasma membranes, in that only certain plasma membrane glycoproteins are shed as part of membrane fragments. The glycoproteins released as non-particulate molecules into the extracellular environment failed to exhibit these quantitative and qualitative differences.     

Biochemical characterization of soluble Tn glycoproteins from malignant effusions of patients with carcinomas

The Tn determinant (GalNAc-O-Ser/Thr) is one of the most specific human tumor markers. In normal cells Tn is a cryptic structure in the peptide core of mucin type O-glycoproteins, and it is detected in an unmasked form in most human carcinomas evaluated by immunohistochemistry. Scarce data are available regarding the characteristics of soluble Tn bearing glycoproteins. We herein report the first comparative characterization of soluble Tn glycoproteins derived from different kinds of human tumors (breast, colon, gastric, ovarian and liver). Considerable heterogeneity was observed in the physicochemical properties of Tn soluble glycoproteins from all the tumor-associated effusions evaluated. In SDS-PAGE analysis Tn glycoproteins from liver and colon effusions migrated as a broad single major component (>500 kDa), while several components of >200 kDa were identified in samples from breast, ovarian, and gastric cancer. The results of perchloric acid (PCA) treatment and CsCl gradient ultracentrifugation indicated that the Tn glycoproteins in effusion fluids correspond predominantly to mucin-like glycoproteins. However, in samples from patients with colon and liver cancer, a fraction of Tn glycoproteins formed part of the immune complexes that precipitated in PCA, suggesting that the anti-Tn immune response in vivo could modify their physicochemical properties. The four apomucins evaluated (MUC1, MUC2, MUC5AC and MUC6) carried Tn epitopes in each of the effusions, indicating that soluble apomucin detection may reflect the abnormal expression of MUC genes inherent to these tumors. Taking together, these results indicate that apomucin expression profile is responsible, at least in part, for the high heterogeneity of soluble Tn glycoproteins, and suggest that the identification of Tn determinant on the different soluble apomucins could be useful for the development of new diagnostic tools as well as to evaluate the anti-tumor immune response in patients with cancer.     

The Molecular Heterogeneity of Nonspecific Cross-reacting Antigen Synthesized by Tumor Cells and Granulocytes

The molecular heterogeneity of nonspecific cross-reacting antigen (NCA) was examined. Metabolically-labeled glycoproteins were precipitated from cell lysates of human tumor cell lines and of normal peripheral granulocytes with antibodies specific for NCA, and analyzed by SDS-PAGE. NCA components synthesized by three tumor cell lines, QGP-1 (pancreas), HLC-1 (lung) and CAOV-2 (ovary) showed slightly different migration patterns on SDS-PAGE, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were all found to be 35K. On the other hand, two molecular species of NCA were identified in normal granulocytes: an 80K mature form derived from a 69K precursor peptide and a 58K mature form from a 41K precursor peptide. Upon SDS-PAGE, the migration pattern of the unglycosylated NCA peptides from tumor cells was affected by the presence of 2-mercaptoethanol, while that of the peptides of granulocytes was not. All the NCAs identified in this study possessed antigenic determinants common to carcinoembryonic antigen as well as those unique to NCA. These results suggest that the molecular heterogeneity of NCA observed thus far resulted from diverse glycosylation of the three fundamental molecular forms of unglycosylated peptides: one with a molecular weight of 35K produced by tumor cells and two with molecular weights of 69K and 41K produced by granulocytes.     

The molecular heterogeneity of nonspecific cross-reacting antigen synthesized by tumor cells and granulocytes

The molecular heterogeneity of nonspecific cross-reacting antigen (NCA) was examined. Metabolically-labeled glycoproteins were precipitated from cell lysates of human tumor cell lines and of normal peripheral granulocytes with antibodies specific for NCA, and analyzed by SDS-PAGE. NCA components synthesized by three tumor cell lines, QGP-1 (pancreas), HLC-1 (lung) and CAOV-2 (ovary) showed slightly different migration patterns on SDS-PAGE, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were all found to be 35K. On the other hand, two molecular species of NCA were identified in normal granulocytes: an 80K mature form derived from a 69K precursor peptide and a 58K mature form from a 41K precursor peptide. Upon SDS-PAGE, the migration pattern of the unglycosylated NCA peptides from tumor cells was affected by the presence of 2-mercaptoethanol, while that of the peptides of granulocytes was not. All the NCAs identified in this study possessed antigenic determinants common to carcinoembryonic antigen as well as those unique to NCA. These results suggest that the molecular heterogeneity of NCA observed thus far resulted from diverse glycosylation of the three fundamental molecular forms of unglycosylated peptides: one with a molecular weight of 35K produced by tumor cells and two with molecular weights of 69K and 41K produced by granulocytes.     

Monoclonal antibodies to glycoproteins of Vinca alkaloid-resistant human leukemic cells

Drug resistance is a major problem in the successful treatment of cancer. Resistance to one drug is often associated with cross-resistance to other anticancer agents. This is commonly seen with the "natural product" anticancer drugs such as the Vinca alkaloids and anthracyclines. In experimental systems, specific changes in plasma membranes characterize this "multiple drug resistance." The most prominent of these is the enhanced expression in several systems of high-molecular-weight glycoproteins ranging from Mr approximately equal to 150,000 to approximately equal to 180,000, the amount of which has been shown to be related to the degree of drug resistance. We report here the production of three monoclonal antibodies that bind preferentially to the surfaces of cultured human leukemic lymphoblasts resistant to the Vinca alkaloid vinblastine. Each antibody recognizes a surface membrane glycoprotein with molecular weights of 180,000 to 210,000. Additionally, two of the antibodies also recognize a second surface glycoprotein with molecular weights of either approximately equal to 155,000 or approximately equal to 130,000. All of these glycoproteins are overexpressed in the alkaloid-resistant cells. While a Mr approximately equal to 180,000 protein has been shown to be associated with multiple drug resistance, the other two glycoproteins have not been described previously in these cells. These antibodies may be useful in studies of the mechanisms of drug resistance, as well as in screening cells from drug-resistant patients for these resistance-associated glycoproteins.     

Combined radiotherapy, chemotherapy, and maltose tetrapalmitate immunotherapy in the treatment of 4'dimethylaminoazobenzene-induced liver cancer

Therapeutic effects of radiotherapy (R), chemotherapy, and maltose tetrapalmitate (MTP) immunotherapy alone and in combinations were tried against 4' dimethylaminoazobenzene (DAB) induced primary liver cancer in Wistar rats in three separate protocols. Rats were fed a low protein synthetic diet containing 0.06% DAB for 90-120 days. Around 90 days, liver cancers developed in all the animals. In the first protocol, animals were either left untreated or treated with cyclophosphamide (Cy), MTP (i.p. or oral) and Cy plus oral MTP. Rats in the MTP (i.p.) group maintained a steady liver weight but neither Cy nor Cy + MTP influenced the survival time or liver weight. In the second protocol, R as well as a 3-drug combination at 2 dose levels were tried alone and with MTP before or soon after cessation of DAB feeding. Survival times were decreased by R and chemotherapy due to combined toxicities of DAB and treatments and were partially restored by MTP. In the third protocol, MTP, R, and Cy were each tried alone and in combinations, 21 days after cessation of 100-day DAB feeding. Increase in survivals were obtained by each treatment, although tumor weight was best controlled by triple R+ Cy + MTP combination.     

Identification of peanut agglutinin binding glycoproteins restricted to Hodgkin's disease-derived cell lines

Peanut agglutinin (PNA) binding glycoproteins from four Hodgkin's disease (HD)-derived cell lines and a variety of cell lines/peripheral blood cells representative of the lymphoid and myeloid lineages were identified by probing nitrocellulose membranes of SDS-PAGE separated NP40 solubilized cellular glycoproteins with [125I]-labelled PNA. The two Hodgkin's cell lines Ho and L428 demonstrated the most heterogeneous glycoprotein profiles each expressing 15 PNA binding glycoproteins, respectively. The two remaining Hodgkin's lines Co and L591 expressed only four glycoproteins each and these were all also commonly expressed by Ho and L428. Comparative analysis with all other cell types studied revealed the expression of five glycoproteins restricted to Ho (gp42, gp40, gp38, gp24 and gp22) and six restricted to L428 (gp180, gp75, gp40, gp38, gp24 and gp22). Four of these, gp40, gp38, gp24 and gp22 were commonly expressed by both Ho and L428. Of cell lines of myeloid lineage studied only the erythroleukemia cell line K562 expressed detectable glycoproteins also expressed by some of the Hodgkin's cell lines (gp110, gp96, gp50 and gp45). Only one glycoprotein, gp20 expressed by Ho was also commonly expressed by normal peripheral blood granulocytes. This limited study has thus succeeded in demonstrating for the range of cell types studied, that some glycoproteins with terminal D-galactose beta (1----3) N-acetyl galactosamine oligosaccharide sequences are apparently restricted to two of the HD cell lines. Moreover, the heterogeneous glycoprotein profiles obtained for the HD cell lines Ho and L428 suggests that galactosylation processes in these two cell lines is aberrant.