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STUDY ON IMMUNOLOGICAL ACTIVITY OF AN OVEREXPRESSED GLYCOPROTEIN ON HEPATOCYTES IN MICE EXPOSED TO N-NITROSODIBUTYLAMINE By Jignasa Mishra Department of Biochemistry Submitted in Fulfillment of the requirement of the Degree of Doctor of Philosophy in Biochemistry Of North-Eastern Hill University, Shillong. 1 ABSTARCT INTRODUCTION Industrial development, which began in the 18th century and continues to this day, has exposed many workers to the hazards of carcinogenic agents. N-nitroso compounds such as NNitrosodibutylamine (DBN) or N-Nitrosodiethylamine (DEN) present industrial occupational hazards to industrial workers. These derivatives have very high degree of cell and tissue specificity depending on systemic distribution of the carcinogen (Mountasano 1982). During nitrosamine induced hepatocarcinogenesis in mice, processes such as gene expression, replication and differentiation are altered in the transformed cell (Pariat and Sharan 1995). Alteration in gene expression in a cell might lead either to the expression of new antigen unique to that tumor or the differential expression of membrane surface glycoproteins. Such antigens are usually called tumor-associated antigen (TAA) and are more useful from the point of view of diagnosis or immunotherapy. Studies carried out by various authors suggest that TAA can serve as an effective target for active immunotherapy against cancer (Raghupati 1996, Goydos et al., 1996, Rodolf et at., 1996). A great majority of human cancers (about 80-90%) are attributable to environmental factors (Benjamin et al., 1990). However, it is not an easy task to eliminate carcinogenic causes from the environment. While modern surgery has significantly reduced the cancer mortality, the use of additional treatment such as radiotherapy and chemotherapy has resulted in no more than 5% reduction in the number of deaths (Benjamin et al., 1990). Therefore, there is a continuing search for better control and preventive methods in order to reduce cancer mortality and related side effects. 2 Tumor immunotherapy is a fast growing area of research and might be the answer to the growing menace that is cancer. One approach in immunotherapy is specific active immunotherapy with antigenic tumor cells which have been rendered non-tumorigenic or extracted tumor associated antigens (TAA). The objective being to stimulate the host immune response against the tumor cells (Ansel and Blangy 1984, Marx 1989). However, the main problem associated with specific active immunotherapy is the generally weak immunogenictiy of TAA (Law 1984). Therefore, a major challenge in tumor immunology is to develop methods that augment potent immune response to TAA. Different methods have been explored. Such as, tumor cells were modified to enhance immunogenicity. This has been accomplished in various ways, for example, by enzymatic unmasking of TAA with enzymes such as neuraminidase (Simmons et al., 1971), by additional immunogenic cell surface determinants (Heicappell et al., 1986), or by rigidification of cell membranes with lipids (Skornick et al., 1984). Immunity against these more immunogenic tumor cells usually cross-reacts with the original tumor cells. Thus, this approach may lead to success. A second approach is to employ immunostimulants as immunological adjuvants. Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum mixed with irradiated tumor cells have been especially studied in the past (Hoover and Hanna 1989). Recently better defined and probably less toxic immunostimulants, such as cytokines and analogs of bacterial products, are under investigation (McCune and Marquis 1990). Antigens on tumor cells are usually also found on normal cells. The expression of such molecules on tumor cells, however, differs from the expression on normal cells, they are usually distributed over the cell membrane, or they are express at an inappropriate phase of oncogenesis, as in the case of fetal antigens on tumor cells in adults. Instead of whole tumor cells, preparation containing extracted TAA of tumor cells (TAA extract) has been explored for active 3 immunization (Fidler 1994). Immunization with purified TAA may be more effective than the whole tumor cells. In the latter case, the relevant TAA may be present at a low density and consequently swamped by other determinants. Isolation and characterization of certain TAA enable the study of immune reactions against highly purified TAA with encouraging results in cancer patients (Hellstrom and Hellstrom 1989). An enhanced expression of a high molecular weight glycoprotein of about 58 kDa on the membrane surface of hepatocytes in mice exposed to DBN has recently been identified by us (Alam et al., 2005). This high molecular weight over-expressed membrane surface glycoprotein TAA is rich in sialic acid residues with significantly high carbohydrate to protein ratio. Therefore, it has great potential as a potent candidate for the development of vaccine and could be used as an effective target for active immunotherapy against cancer. In the present investigation, an attempt was made to study the immunological activity of this over-expressed high molecular weight (~58 kDa) glycoprotein TAA for its suitability to use as a potent candidate for vaccine development. METHODOLOGY Carcinogenesis was induced in Swiss albino mice by intra venous administration of DBN (10 mg/kg body weight) as has been reported earlier (Alam et al., 2001, Alam et al., 2005). Enzyme markers (gamma glutamyl transpeptidase, acetylcholine esterase and glutathione transferase) activities, Glutathione level and the histological examination of liver tissue were the parameters used to follow up cancer induction in DBN-treated mice. Sham-treated, age matched normal mice will serve as control. 4 The procedure described by Liao et al., (1984) has been followed to extract glycoprotein from liver tissues of both the normal as well as DBN-treated mice. This method selectively extracts membrane surface glycoproteins. The crude TAA extracts from normal and treated mice were subjected to SDS-PAGE analysis to resolve and identify the tumor-associated antigen (TAA). Molecular weight marker was used to determine the approximate molecular weight of desired TAA. Ion-exchange and gel filtration chromatography and preparative SDS-PAGE techniques were employed to purify and determine the molecular weight of TAA. Purified TAA (58 kDa glycoprotein) in different formulations was tested for its immunogenicity in mice. Immunogenicity, after enzymatic unmasking of purified TAA with enzyme neuraminidase was also studied and a comparison was made for better immunogenicity of these two forms of TAA. ELISA was used to determine the concentration of antibody against TAA in the serum sample of immunized and normal control mice. Both positive and negative controls were used in the study. RESULTS Hepatocarcinogenesis was induced successfully in mice by aqueous administration of DBN intravenously at weekly dose of 10mg/kg body weight for the period up to 16 weeks. DBN-exposed animal appeared to be healthy as they did not show any sign of lethargy or sluggishness or enlargement of the liver. Successful induction of hepatocarcinogen was observed as liver excised from DBN-treated mice showed nodule formation which was pale in color also some livers of treated mice showed hardening, swelling and pigmentation of the tissue when compared to age- matched normal control mice. 5 The elevated level of liver marker enzymes activities (GGT, AChE), increase level of GSH were also observed in DBN-treated mice. The elevated level of GGT in DBN-exposed mice signifies hepatocellular transformation since GGT activity has been found to increase in many growing tissues, aging cells and differentiating tissues. The release of more enzyme acetylcholine esterase into supernatant signifies that DBN has induced membrane damage and support the development of a cancerous situation in hepatocytes. Increase in GSH level in liver cells of DBN-exposed animals may likely be due to a rise in GGT activity. Increase level of GGT has been correlated with modulation in cellular GSH level. Lower level of GST activity was significantly noticed in DBN-exposed mice as compared to normal control mice. The decrease in GST activity in DBN-exposed animals signifies that it leaves the cell vulnerable to these electrophiles & other reactive species. It also suggests that depletion of cellular protection may be an important event in initiation of carcinogenesis. The levels of SGOT and SGPT enzyme in DBN-exposed mice showed significant rise in their activities as compared to normal control mice. These significant changes in treated mice signify that DBN induction has caused liver damage to some extent. Histological sections of DBN-exposed mice showed several morphological changes like the liver cells of treated mice were less well differentiated than the normal control, they had irregular outline with an increased in number of nucleus-to-cytoplasmic ratio. The cells were loosely packed in the tissue showing their loss of contact with the neighboring cells. The 1-butanol extract of liver tissue of DBN-exposed mice showed differential expression of proteins as compared to that of normal control mice. The SDS-PAGE resolution showed the presence of an over-expressed glycoprotein of 58kDa molecular weight in DBN- 6 exposed mice as compared to normal control where this protein was either present in trace amount or disappeared. This glycoprotein was identified as the TAA in our study. Successful attempt was made to purify this TAA in stages. The first purification step carried out was anion-exchange chromatography. The TAA was eluted from the bound fraction of anion-exchange column, indicating that this protein was an anionic glycoprotein. This anionic nature of TAA could be due to the presence of negatively charged sugar residues. The second purification stage involved purification of TAA from preparative SDS-PAGE gels. The TAA obtained by this method was significantly high, pure and homogeneous. After purification of TAA, Immunization against TAA in crude form and after unmasking of TAA with neuraminidase enzyme was carried out and comparison was made for immune-reactivity between these two forms of TAA. Immunological study was carried out to study the immunogeneicity of TAA both in crude and de-glycosylated forms. De-glycosylation test carried out on purified TAA resulted in a decrease in molecular weight of TAA as the de-glycosylated enzyme used here removes terminal proteins or sialic acid. After treatment of TAA with neuraminidase resulted in two protein bands of decreasing molecular weight which were approximately 45 kDa and 38 kDa as resolved in SDS-PAGE gels. These partially purified TAA was tested for its immunoreactivity and was found to be highly immunogenic. A high level of anti -TAA antibodies were detected in mice upon immunization and were evident from its anti-TAA titres determined by ELISA as compared to mice immunized with SDS-PAGE gel homogenate prepared from crude TAA thus indicating that 7 de-glycosylated TAA was more immunogenic in mice as compared to purified crude TAA extract. The glycoprotein (58kDa) was found to be highly immunogenic as was evident from the high and persistent level of antibodies raised upon immunization. The immunogenicity of glycoprotein (58kDa) was further enhanced significantly upon treatment with neuraminidase. Thus the over-expressed high molecular weight (58kDa) glycoprotein TAA could eventually be used as a potential candidate for vaccine development to induce immune response against cancer in order to counter regression. REFERENCES: Alam A, Singha LI, Singh V, (2005) Molecular characterization of tumor associated antigen in mice exposed to a hepatocarcinogen. Mol. Cell. Biochem., 271, 177-188. 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