Download Jignasa Mishra Department of Biochemistry Submitted in Fulfillment

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.
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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.
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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
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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.
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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.
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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-
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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
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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.
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