Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Detection of anti-Hu antibodies, a possible key to early diagnosis of small cell lung cancer
(USC Thesis Other)
Detection of anti-Hu antibodies, a possible key to early diagnosis of small cell lung cancer
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
INFORMATION TO USERS
This manuscript has been reproduced from the microfilm master. U M I films
the text directly from the original or copy submitted. Thus, some thesis and
dissertation copies are in typewriter face, while others may be from any type of
computer printer.
The quality of this reproduction is dependent upon the quality of the
copy submitted. Broken or indistinct print, colored or poor quality illustrations
and photographs, print bleedthrough, substandard margins, and improper
alignment can adversely affect reproduction.
In the unlikely event that the author did not send UM I a complete manuscript
and there are missing pages, these will be noted. Also, if unauthorized
copyright material had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by
sectioning the original, beginning at the upper left-hand comer and continuing
from left to right in equal sections with small overlaps.
Photographs included in the original manuscript have been reproduced
xerographically in this copy. Higher quality 6” x 9” black and white
photographic prints are available for any photographs or illustrations appearing
in this copy for an additional charge. Contact UM I directly to order.
Bell & Howell Information and Learning
300 North Zeeb Road, Ann Arbor, M l 48106-1346 USA
800-521-0600
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DETECTION OF ANTI-HU ANTIBODIES, A POSSIBLE KEY TO
EARLY DIAGNOSIS OF SMALL CELL LUNG CANCER
Copyright 1999
by
Jeffrey An-Pang Tsou
A Thesis Presented to the
Faculty of the Graduate School
University of Southern California
In Partial Fulfillment o f the
Requirements for the Degree
Master of Science
(Biochemistry and Molecular Biology)
August 1999
Jeffrey An-Pang Tsou
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
UMI Number: 1397649
UMI
UMI Microform 1397649
Copyright 2000 by Bell & Howell Information and Learning Company.
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.
Bell & Howell Information and Learning Company
300 North Zeeb Road
P.O. Box 1346
Ann Arbor, Ml 48106-1346
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
UNIVERSITY O F SO U T H E R N CALIFORNIA
TH E GRADUATE SC H O O L
U N IV ERSITY PARK
LOS A N G ELE S. C A LIFO R N IA ( 0 0 0 7
This thesis, 'written by
!& £ £ £ lU & 3 jS e!± ______________________
under the direction of h„ 15. Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
,.„1^3SJCSJC...QjE_Sclence
Date___
THESIS COMMITTEE
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table of Contents
Chapters pages
1. Abstract 1-2
2. Introduction 3-9
3. Materials and Methods 10-17
4. Results 18-30
5. Discussion 31-35
6. Bibliography 36-40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
List of Figures
Figure Title
1. Cloning a Biotinylatable Tag into the
C-terminus of Hel-Nl
2. Cloning a Biotinylatable Tag into the
N-terminus of Hel-Nl
3. Protein Induction and Purification of
Hel-Nl with the Biotinylatable Tag
at the C-terminal
4. Protein Induction and Purification of
Hel-Nl with the Biotinylatable Tag
At the N-terminus
5. Low Amounts of Biotinylatation with the
C-terminal Biotinylatable Tag
6. Successful Biotinylatation of Hel-N 1
Containing the Biotinylatable Tag at
The N-terminus
7. Determining the Amount of Protein Biotinylated
8. Ability of Hel-Nl to be Bound
by Anti-Hu Antibodies
pages
19
20
21
23
24
26
28
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Abstract
Of all cancer deaths that occur each year, lung cancer accounts for 28% of them,
making it the leading cause of cancer mortality. 85% of lung cancer cases can be
attributed to smoking. Lung cancer is a heterogeneous disease that can involve many
different cell types. The four major types of lung cancer are squamous carcinoma, adeno
carcinoma, small cell lung carcinoma (SCLC) and large cell lung carcinoma. Small cell
lung carcinoma is the most aggressive and metastatic.
Paraneoplastic encephalomyelitis sensory neuronopathy (PEM/PSN) is a
neurological disease that is found in association with small cell lung carcinoma. This
disease can be characterized by dementia, neuronal degeneration, and destruction of the
nervous system. Patients who exhibit PEM/PSN have been shown to have a high titer of
certain antibodies called anti-Hu antibodies. The Hu proteins against which the
antibodies are directed are produced in all small cell lung carcinomas. It has been
hypothesized that the immune response to the Hu proteins present in SCLC is involved in
PEM/PSN neurological disease. The immune response is thought to attack the Hu
proteins that are normally expressed in the nervous system thus resulting in this
neurological disease. Hu proteins are a family of 35-40 kD RNA binding proteins, three
of which (HuD, HuC, and Hel-Nl) are normally expressed in the nervous system. It is
surprising that these proteins are also produced by small ceil lung carcinoma.
Although the percentage of SCLC patients who have high titers of anti-Hu
antibodies is less than 1%, western blot analysis has shown that 16% of patients have
detectable anti-Hu antibodies. The presence of these highly specific anti-Hu antibodies
l
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
in SCLC may offer a method for early detection of the cancer. This however, would only
be useful if a higher percentage of patient sera show detectable anti-Hu antibodies. To
determine if this maybe the case, my goal was to use a more sensitive method, surface
plasmon resonance to detect anti-Hu antibodies. This method uses a BIAcore machine.
My project was aimed at determining whether cloned Hu proteins coupled to a BIAcore
chip can be used to detect anti-Hu antibodies in patient sera.
The first objective was to develop a system of coupling the Hu protein to a
BIAcore sensor chip through biotin avidin interactions. A biotinylatable tag to which a
single biotin can be added was cloned into either the N terminal or the C-terminal end of
the Hel-Nl cDNA expression vector. Protein induction was performed in E.coli. The
HEL-N1 vector with a biotin tag at the N-terminal successfully gave rise to protein
biotinylated which was with 97% efficiency, whereas the tag at the C-terminus yielded
insufficient amounts of biotinylated protein. Biotinylation appears to have no affect on
the epitope of the protein to which the anti-Hu antibodies would bind. This is an effective
stepping stone in the climb towards early detection of lung cancer.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Introduction
Lung cancer is the leading cause of cancer mortality in men and women in the
United States (49). By the end of this year, it is anticipated there will have been one
million deaths attributed to this disease throughout the world. Lung cancer accounts for
28% of all cancer deaths each year and in some countries (such as the United States) has
surpassed breast cancer to become the leading cause of cancer deaths in women as well
as in men (8). It has been estimated that 85% of lung cancer deaths can be attributed to
smoking. In the United States, lung cancer has displaced coronary heart disease as the
leading cause of excess mortality among smokers (49).
Lung cancer is considered a heterogeneous disease, because of the variety of cell
types involved. There are four major types of cells that make up lung cancer; squamous
carcinoma, adeno carcinoma, small cell lung carcinoma (SCLC) and large cell lung
carcinoma. The difference in biological behavior in the types of cells is another example
of its heterogeneity. SCLC makes up around 16-23% of the lung cancers and is the most
aggressive and metastatic of the cancers. SCLC derives its name from the tumor cells
that are characterized by small size. It has a round to fusiform shape, scant cytoplasm,
finely granular nuclear chromatin, and absent or inconspicuous nucleoli.
There are constant developments in science that attempt to improve the detection
and treatment of lung cancer. The most logical and simplistic strategy that has been
considered is the notion of early detection. It has been shown that surgical resection is
the only approach that offers a potential cure, but it is effective only for localized diseases
(59). Removal of the localized cancer can prevent the further growth and metastasis of
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
the cancer. The growth rate and spread of the tumor can vary, thus the timing, selection
of the type of detection and the mode of intervention, can be difficult. Timing is thus a
key factor in early detection. Studies have shown that most patients who have surgical
resection of early stage tumors tend to achieve long-term survival (22).
Cigarette smoking is estimated to be the cause of 85% of lung cancers, however
genetic predisposition also plays a role (37,49). The most effective way to cure lung
cancer is prevention of exposure to cigarette smoke. Studies have shown that initiation
and promotion of lung cancer are due to a series of genetic events, such as point
mutations, chromosomal abnormalities, gene amplification, and altered gene expression
(50). An increased risk factor that is involved in genetic predisposition of lung cancer is
the function of Glutathione S- transferase Ml (GSTM1). This enzyme is involved in
detoxication of many carcinogens, such as polyaromatic hydrocarbons as found in
cigarette smoke. It has been shown that in 30%-55% of individuals, the GSTM1 enzyme
is missing due to deletions in both copies of the GSTMl-gene (37). Those individuals
may be more susceptible to lung cancer than those who have the GSTM1 enzyme. Thus,
there are environmental and genetic risk factors, many of which remain to be
characterized that put certain populations at high risk for developing lung cancer. A
successful tool for early detection, could be crucial to prevent many lung cancer deaths.
A neurological disorder that was found associated with SCLC is paraneoplastic
encephalomyelitis sensory neuronopathy (PEM/PSN). It is characterized by dementia,
cerebellar degeneration, brain stem encephalopathy, myelopathy, and sensory neuropathy
(12,55). Research suggests it is caused by an immune response that is primarily directed
against the tumor and certain proteins (Hu proteins) it produces (51). It is thought that
4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
the anti-Hu antibodies that were originally directed against the tumor cells are
misdirected against similar antigens expressed in the nervous system (55).
Hu proteins are family of proteins that are 35-40 kD RNA binding proteins, which
contain three highly conserved RNA recognition motifs that are referred to as RRM1,
RRM2, and RRM3. Four different genes in the Hu family are known to yield several
highly conserved products: HuD, HuC, HuR and Hel-Nl. HuR, the least conserved of
the Hu family of proteins is ubiquitously expressed in all cells while HuD, HuC, and Hel-
Nl are expressed in neurons. Hu proteins were discovered originally because they were
the targets of antibodies found in patients with PEM/PSN associated with SCLC (33).
Patients who have PEM/PSN have a high titer of the anti-Hu antibodies. Anti-Hu
antibodies from patients have been shown to recognize with HuD, HuC, and Hel-
Nl expressed by neurons and SCLC (40,25).
The Hu proteins are homologous to the Drosophila protein ELAV (embryonic
lethal abnormal visual system), to which they are 86-90% identical. ELAV is required
for normal development and maintenance of the fly nervous system. (40) Flies that
contain mutations in ELAV do not develop beyond the embryo. The Hu proteins display
a pattern of expression during vertebrate neurogenesis similar to ELAV. Like ELAV, Hu
proteins are assumed to take part in the maintenance and development of the nervous
system.
The Hu gene family was cloned from mouse and Xenopus and shown to have
ordered features of expression (23,43). Specific Hu genes are expressed in a hierarchy
during early neurogenesis whereas Hel-Nl was induced first in very early postmitotic
neurons (43). Past studies have shown that Hel-Nl is neuron specific and is involved in
5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
development of the central nervous system (21). Evidence of Hel-Nl being primarily
neuron specific came from the characterization of the DNA flanking the 5’ end of Hel-Nl
(30). The promoter elements suggested they were involved in transcription enhancement
and were likely to contribute to the neuronal specificity of Hel-Nl mRNA expression.
This supports the idea that Hel-Nl is neuron specific and the detected proteins in SCLC
are not endogenous. Hel-Nl was also shown to be able to bind to mRNA poly A tails
thus involving it in a post tanscriptional level of gene regulation. It is suggested that the
role of Hel-Nl acts as a transcription factor to act neuronal differentiation and
development.
Which of the Hu proteins are expressed in SCLC? Early studies had indicated
that only HuD was expressed in SCLC (40). However, recent PCR analysis has shown
that Hel-Nl was also expressed in SCLC (31). By means of RT-PCR, the use of specific
primers derived from the 5’ end of the gene has shown that Hel-Nl is expressed by small
cell lung carcinoma. Hel-Nl was expressed in SCLC. Work from the Laird-Offringa
lab has shown that SCLC lines can express different constructions of Hu proteins
(manuscript in prep). All SCLC lines express HuR and Hel-Nl mRNA. The presence of
Hel-Nl in every cell line, cross reacts with anti-Hu antibodies and it’s early neuronal
expression, make Hel-Nl a good candidate as a preliminary target to detect anti-Hu
antibodies in patient sera.
Through ELISA, immunohistochemistry, and sensitive quantitative western blot
analysis, anti-Hu antibodies were detected in 16% of patients with SCLC without
PEM/PSN disorder (25,11). These assays showed low titers of anti-Hu antibodies in these
patients compared to high titers of anti-Hu antibodies in patients with rare cases of SCLC
6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and PSN/PEM. The ability to better and more sensitively detect anti-Hu antibodies in
serum of patients with SCLC could provide a new method of early detection for SCLC.
One assay that may be considered is through the use of BIAcore.
BIAcore is used to study interactions between biomolecules in real time (45). The
BIAcore has been successfully used to study a variety of interactions, for example, the
kinetics of protein, DNA, and RNA binding. There are various elements involved in the
use of BIAcore such as, an optical phenomenon, a stationary interactant and a mobile
interactant. Surface plasmon resonance is the optical phenomenon that is derived from a
light source that is a high efficiency near infared light emitting diode. The light is
directed on to a sensor chip in a wedge-shaped beam, creating a range of fixed incident
angles. All the angles of light are reflected by the surface chip except one, which is
absorbed in the surface chip due to the surface chip’s composition. A computer is used to
record and calculate with a high degree of accuracy, the missing angle. The information
recorded by the computer visualizes the changes in the mass concentration of molecules
in the assay. This allows a real time detection of binding with various macromolecules
involved in the assay.
The BIA assay involves a mobile interactant, and a stationary interactant that is
located in a flow cell of the assay machinery. The stationary interactant is immobilized
on the sensor surface while a solution containing the mobile interactant is allowed to flow
continuously over the sensor surface in the flow cell. In the flow cell, the target molecule
on the stationary interactant is allowed to be bound by the molecule in the mobile
interactant. The binding of the stationary and mobile interactant creates a change in
surface chip concentration and is recorded by the computer. The stationary interactant is
7
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
bound to the sensor surface or sensor chip, which is a slide containing a thin layer of gold
coated on one side. Gold is chosen because of its good surface plasmon resonance and its
chemical inertness. The gold coating on the chip is covered with a covalently bound
carboxymethyl dextran. The matrix serves to allow covalent immobilization of
biomolecules using well characterized chemistry. It increases sensitivity by increasing
the binding capacity of the surface and provides a hydrophilic environment suitable for
most interactions of biological interest. Lastly, it provides a very low degree of non
specific binding to the surface (45). In our assay the immobile interactant is the Hu
protein and the mobile interactant is the antibodies in the patient sera.
A strategy that is common in coating the molecule of choice to the matrix of the
chip is by biotin avidin interactions. The carboxymethyl dextran portion of the matrix is
chemically coupled to avidin while the molecule of choice is biotinylated. The
extraordinary affinity that biotin and avidin have for one another where Kn= 101 5 M '1 is
of major interest and used in many assays. Avidin is a protein that is found in egg whites
and was discovered from intensive nutritional investigations into the vitamin B complex
(5). Nutritional deficiency in rats was discovered when avidin, a minor constituent of egg
white formed an extremely stable noncovalent complex with B vitamin biotin. The role
of avidin, which is only rarely expressed, is described as a scavenger, inhibiting bacterial
growth. A close relative streptavidin is very similar to avidin and is expressed in a
species of streptomyces. A distinctive feature of the avidin biotin complex is that avidin
has four binding sites for biotin. The rationale in using the avidin biotin system is based
on the idea that if one chemically modifies any biologically active compound with biotin,
8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
the biological and physicochemical properties of the biotin- modified molecule will not
be changed significantly (58).
The first step in determining whether the BIAcore could be used to detect anti-Hu
antibodies would be to devise a reproducible and stable way to coat the protein onto the
chip. The initial objective of my project is to biotinylate cloned Hu proteins and couple
them to sensor chips through biotin avidin interactions. This would be achieved by
cloning a biotinylatable tag into a bacterial Hel-Nl expression vector. A single biotin
would then be placed on the bitotinylatable tag in vivo during expression in E.coli. The
subsequent goals of this project are to coat the biotinylated protein onto the chip and to
determine its ability to bind to serum antibodies. This thesis describes my work on the
first aim of this project.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Materials and Methods
C-terminal biotinylatable tag vector construction
A biotinylatable tag was cloned into the C-terminus of the Hel-Nl cDNA in a 6.2
kilobase (kb) pET- Hel-Nl vector (F622) with a pBR322 backbone. The sequence of the
C-terminal biotinylatable tag was GGGLNDIFEAQKIEWH (obtained from
Avidity). Oligonucleotides used for the cloning were ordered from Fisher Scientific
Genosys. The C-terminal tag was cloned into the Not I site that is located at the C-
terminus of the Hel-Nl gene. The tag was specially designed to regenerate a Not I site at
the 3’end of the tag only.
For the cloning of the C-terminal tag, 5 ug of F622 Hel-Nl vector was
digested with Not I overnight at 37°C. The vector was run on a 1% agarose gel for 1 hour
at 120 volts and purified by Qiagen quick spin gel purification kit. The oligonucleotides
used for the biotinylatable tag were reconstituted in water to 100 pmol/ul and were
annealed at 850 C for 3 minutes then cooled to room temperature. 10 pmol of the
annealed oligos was ligated into 14 ng of vector with a ratio of 1:10 molar excess tag to
vector in a ligation mix containing lOx ligation buffer (Pharmacia). The ligation was
incubated at room temperature for 2 hours. Following incubation, 4 ul of ligation mix
was transformed into DH12S competent cells, heat shocked at 37° C for 50 seconds and
placed on ice. 200 ul of LB was added to the cells and incubated at 37° C for lhour then
plated on ampicillin LB agar plates and incubated overnight at 37°C. Colonies that grew
from the overnight plating were used in a PCR screening to determine the success of the
10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
ligation and correct orientation. The colonies were picked with a toothpick, resuspended
in 50 ul of water and prepared for PCR screening. The PCR reaction mix contained 10
mM of each nucleotide (dATP, dCTP, dGTP, dTTP), 100 pmol of each primer and I OX
PCR buffer (Promega). The 5’ primer, used to primer upstream of the insert was
(CATGCCATGGCCTATGGCG TAAAGAG) and ordered from Genosys. A 3’
primer with the sequence CAACTTTCAACAGTCTATGC was used to prime the
downstream of the insert. The PCR products were digested with Not I to determine the
correct orientation of the biotinylatable tag. A clone with the correct insert orientation
was grown up in 500 ml LB with ampicillin overnight and DNA was prepared using
Qiagen Maxi prep kit.
N-terminal biotinylatable tag vector construction
The site used to clone the biotinylatable tag into the N-terminus of the Hel-Nl
cDNA was an Nco I site, which resides at the Hel-Nl ATG. The N-terminal
biotinylatable tag contained the amino acid sequence M S G L N D I F E A Q K I E W
H G A P (obtained from Avidity). Oligonucleotides used for cloning the tag into the
vector were designed and ordered from Genosys. The design of the oligonucleotides was
such that a Nco I site would be regenerated only at the 3’ end of the tag. The oligos were
reconstituted in water to 100 pmol/ul, annealed at 85°C for 3 minutes and cooled to room
temperature
There are two Nco I sites in the coding region of HEL-N1, one at the N- terminus
(ATG) and the other near the C-terminus. The biotinylatable tag was cloned into the Nco
U
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
I site at the N-terminus of the coding region (fig. 1) in a two step procedure. 20 ug of the
F622 vector was digested with the restriction enzyme Nco I overnight at 37°C. The
digested vector, which yielded two fragments, was separated on a 1% agarose gel for 1.5
hours at 120 volts. The two bands were isolated and gel purified using the Gibco Gel
Purification kit.
The N-terminal biotinylatable tag was ligated into the vector with a ratio of 1:10
molar excess tag to vector in a ligation mix containing 10X ligation buffer (Pharmacia).
Following a 2-3 hour incubation at room temperature, the ligation reaction mix was
transformed into DH12S competent cells. For the transformation reaction, 4 ul of
ligation mix was added to 20ul of competent cells, and heat shocked at 37°C for 50
seconds. The cells were put on ice and 200 ul of LB was added, then incubated at 37°C
for 1 hour. The transformation was plated on ampicillin LB agarose plates and incubated
overnight at 37°C. To identify a successful ligation products, a PCR screening was done
with 10X buffer (Promega), lOmM of each nucleotide (dATP, dCTP, dTTP, dGTP), and
100 pmol of each primer. The 5’ primer with the sequence TTGTTTAACTTTAAGAA
GGAG, the 3’ primer with the sequence ATTGAGGCGGCCGCCGGACGGGCAT
ATGAGACCTT TATGGTT were ordered from Microchemical Core Facility.
A correct clone containing the biotinylatable tag was chosen and grown up in a
500 ml inoculation overnight and prepared for a maxi prep. The vector was prepared for
the insertion of the missing Nco I fragment of the coding region by Nco I digestion. The
linearized vector was isolated and gel purified by Gibco quick spin gel purification kit.
Prior to the ligation of the Hel-Nl Nco I coding fragment, the vector with the N-terminal
biotinylatable tag was de-phosporylated. Calf Alkaline Phosphatase from Pharmacia
12
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Biotech was added to the vector and incubated for 10 minutes at 55° followed by an
additional amount of Calf Alkaline Phosphatase and further incubation at 37° for 10
minutes. To inactivate the phosphatase, the vector and enzyme were incubated at 65°C
for 1 hour. The Hel-Nl gene fragment was ligated into the vector containing the N-
terminal biotinylatable tag with a ratio of 1:10 molar excess insert to vector and incubated
at 16°C overnight. The ligation was transformed into DH12S competent cells, plated on
ampicillin LB agarose plates and incubated at 37°C overnight as described earlier.
Mini preps were prepared to determine if ligation was achieved by selecting
colonies and inoculating them in 2ml of LB plus ampicillin overnight. The next day cells
were centrifuged at 14K rpm for 1 minute and resuspended in 100 ul quick mix (0.05 M
glucose, 0.025 M Tris pH 8.0 and 0.01 M EDTA). A 200 ul lysis mix was added (0.2 N
NaOH, and 1% SDS) and samples were set on ice. 150 ml 3M Kac pH 4.8 was added
and samples were incubated on ice for 5 minutes. Lysates were centrifuged at 14K. rpm
for 5 minutes and the supernatant containing the DNA was added and Phenol extracted.
The DNA was ethanol precipitated and resupended in 50 ul of TE (10 mM Tris pH 8, 1
mM EDTA) with RNAse (lOmg/ml). The mini preps were digested with Nco I to
determine if the ligation was successful. The orientation of the ligated Hel-Nl gene was
determined to be correct by digestion with Xba I and Hind III restriction enzymes.
Protein induction and purification
The protein purification and induction procedure was similar for both vectors
containing either the N-terminal biotinylatable tag or the C-terminal tag. pBirAcm,
13
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
received from Avidity, is a plasmid with a pACYC184 backbone. It contains the E.coli
Bir A gene which is a biotin ligase. The biotin tag clone and the pBirAcm vector, both
which are IPTG inducible, were co-transformed into BL-21 DE3 competent cell lines
similar to the transformation procedure stated earlier. The transformation was plated on
agar LB ampicillin and chloramphenicol plates to select for the Hel-Nl and Bir A
plasmids respectively, and incubated at 37°C overnight.
The next day colonies were chosen and inoculated for overnight growth in a 100
ml of LB with ampicillin and chloremphenicol. The following day the cells were diluted
1:5 and allowed to grow for 1 hour at 37°C. Readings were taking using the
spectrophotometer at 600 nm. The cells were ready for induction when the O D < so o = 1 0.
Cell were induced with the addition of 48mg/ml of IPTG (Isopropyl-B-D-
Thiogalactoside) and incubated for 3 hours at 37°C with shaking. Following the
incubation, the cells were pelleted at 5000 rpm for 10 minutes and resuspended in
sonication buffer, (10 mM Tris pH 8.0,150 mM NaCl, 0.5% Triton X 100). The cells
were sonicated (sonicate at pulse 3 for 30 seconds, cool between sonication for 1 minute,
repeat procedure 4X) and pelleted for 10 minutes at 13,000 rpm in microfuge. The
supernatant was isolated and saved for protein purification. The supernatant was added
to nickel beads from Qiagen for a batch-wise protein purification. The protein was bound
to nickel beads in suspension through the His tag that had been previously engineered on
the C-terminal end of the protein (Dr. Laird-Offringa). The beads were washed with
sonication buffer containing 5 mM imidazole. Protein was eluted with elution buffer
containing increasing concentrations of imidazole: 50,100, 150,200,250 mM in wash
buffer (wash buffer = sonication buffer with 10% glycerol).
14
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Detection of biotinvlation
A western blot transfer was done to determine if the protein was biotinylated. 10
ul fractions of the various protein elutions were run on a 10% polyacrylamide SDS
denaturing gel. The size of the expected protein is 40-45 kD and can be seen on the
Coomasie blue stained gel. (Fig. 4) 10 ul of each eluted sample was added to 10 ul of 2x
lysis mix (1M Tris pH 6.8,20% SDS, 1/20 vol glycerol, 1/10 vol B-mercaptoethanol,
Bromophenol blue saturated, H20). The gel was transferred to nitrocellulose membrane
from (MSI). The membrane was blocked with TBST (10 mM Tris pH 8.0, 150 mM
NaCl, 0.05 % Tween 20) with 3% BSA for 2-3 hours. The membrane was probed with
extravidin horseradish peroxidase (HRPO) from Sigma, and incubated for 30 minutes.
After incubation the membrane was washed 6 times for 5 minutes each in TBS (10 mM
Tris pH 8.0, 150mM NaCl). The membrane was then developed with Super Signal from
Pierce for 5 minutes following the manufacturers recommendations. The membrane was
then exposed to X-ray film for 1,2, 5, 10 seconds.
Determination of the percentage of protein biotinylated
In order to determine the percentage of protein that was biotinylated, streptavidin
paramagnetic beads (Dynal) were used. The protein that is biotinylated can be bound to
the beads, and the paramagnetic nature of the beads allows the isolation of the protein.
The unbiotinylatated protein was washed away by sonication buffer (10 mM Tris pH 8.0,
15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
150 mM NaCl, 0.5% Triton X 100). A comparison was made between the amount of
protein present before and after the addition to the beads. Bead binding involved the
addition of lug of protein to 50ul of paramagnetic avidin beads. The beads were
incubated at 4° for 1 hour on a rotator.
After the incubation, the beads were separated by a magnet and the supernatant
was collected. The supernatant was the amount of protein not bound to the beads and was
considered unbiotinylated. The beads were then washed three times in sonication buffer.
The remaining beads and their bound protein were boiled at 100°C for 5 minutes in 2X
lysis mix, releasing the protein into the supernatant. The supernatant was collected and
the amount of protein biotinylated was determined. The supernatant from the boiled
beads were diluted 2 fold in a serial dilution from IX to 1/32X. All samples including
initial amount of protein added, non bound, and serially diluted were run on Coomasie
gel.
Ability of protein to bind antibody in native form
In order to determine whether the addition of a biotin affects the ability of the protein to
be bound by anti-Hu antibodies, an immunoprecipitation assay was performed. A ratio of
1:1:10 antibody to antigen to protein G was used. Antibody and antigen were bound at a
concentration of lmg/ml each in binding buffer (25 mM Hepes pH 7.5,300 mM NaCl,
0.1% Np40,0.25% Tween 20) for 1 hour at 4°C. Protein G agarose (Sigma) was used to
pull down the antibodies. The complex was washed 3 times in wash buffer, (25 mM
Hepes, 600 mM NaCl, 0.1 % NP40,0.25% Tween 20) for 10 minutes each and
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
centrifuged at < lOOOg in a microfuge. The beads were harvested and 2x lysis mix was
added, samples were boiled for 5 minutes at 100° C and loaded onto a 10%
polyacrylamide SDS protein gel. The gels were made in duplicate, one for Coomasie
staining and the other for western blot transfer.
The protein was transferred to a nitrocellulose membrane and blocked overnight
with TBST containing 3% BSA as described above. A murine antibody to the C-terminal
Myc tag was used as a primary antibody to probe the membrane. The antibody was
diluted 1:5000 in TBST containing 3% BSA. Following incubation at room temperature
for 2 hours, the membrane was washed 3 times for 10 minutes with TBST, 0.3% BSA.
The secondary antibody, a biotinylated goat anti-mouse antibody diluted 1:20,000 in
TBST, 3% BSA, was added and the blot was incubated at room temperature for 1 hour.
The membrane was washed 3 times for 10 minutes with TBST 0.3% BSA. ExtrAvidin
horseradish peroxidase conjugate from Sigma was diluted 1:5000 in TBST and incubated
with the blot at room temperature for 30 minutes. The membrane was washed 5 times for
6 minutes in TBS and substrate Super Signal (Pierce) was added and incubated for 5
minutes.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Results
Construction and Analysis of the clone with the C-terminal and N-terminal biotinylatable
tag
The biotinylatable tag was inserted into the C-terminus of the Hel-Nl cDNA, as
shown in figure 1. The new bacterial expression vector with the C-terminal
biotinylatable tag was prepared for protein induction. Figure 2 shows the procedure
followed for ligation of the biotinylatable tag into the N-terminus of the pET-Hel-Nl
vector.
Protein induction and purification
Protein induction involved the co-transformation of Hel-Nl with the
biotinylatable tag inserted in either the N or C terminus of the cDNA and a Bir A vector
(for the expression of biotin Iigase) into BL21 DE3 competent cells. IPTG will induce
expression with the Bir A gene, as well as of the T7 polymerase. The T7 polymerase will
activate the T7 promoter during the expression of Hel-Nl.
Protein was induced using the Hel-Nl vector containing the biotinylatable tag
located at the C-terminus. Purification of the protein involved elution buffers that
contained increasing concentrations of imidazole ranging from 50 mM to 250 mM. The
Hu family of proteins are 35-40 kD in size. Figure 3 shows the migration of the protein
1 8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
Nco1
T*
\
Nco1
I N o ll s to P coclon
p w m a m |[T im l
/
/
D igest w ith Not1 (F 622)
Ncol
Ne° 1 . . . . I Noll ,
' I | j j T 7 - \ kn I
Biotinylatable Tag
In s e rt T ag
Ncol
Nco1
I |^BtHaW *llS5n
Not1
Figure 1: Cloning a Biotinylatable tag into the C-term inus of Hel-Nl
The pET-Hel-Nl vector was digested with Not 1 to prepare for cloning.
The 60 bp biotinylatable tag was designed to regenerate the 3’ Not 1 site
only after cloning. The biotinylatable tag was cloned into the Not I site at
the C-terminus o f the Hel-Nl vector upstream o f the His and Myc epitope
tag.
ca
His Tag
Myc Tag
Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
Ncol
£L3JTrrtE2iil
1. Digest with Ncol
Blotlnylatabla Tag
Ncol
1. Red/gest with Ncol
Biotinylatable Tag Ncol
2. Re-Insert Hel-Nl
Ncol Not1 / Ncol Ncol Not1 '
/
/
C Z 3
His Tag
za Myc Tag
Figure 2: Cloning a biotinylatable tag into the N-terminus of Hel-Nl
The biotinylatable tag was cloned into the Nco I site at the N-terminus of the coding region
in a two step procedure. The pET-Hel-Nl vector was digested with Nco 1 yielding a 1 Kb
fragment containing the Hel-Nl gene and a 5 Kb fragment containing the rest of the vector.
The 57 bp biotinylatable tag was cloned into the Nco 1 site at the N-terminus (ATG) of the 5
Kb vector. The tag was designed to regenerate the 3’ Nco 1 site only. The 5 Kb vector
containing the biotinylatable tag was digested with Nco 1. The 1 Kb Hel-N 1 gene was cloned
back into the vector containing the biotinyltatable tag.
— 244 Kd
— 80 Kd
— 42 Kd
— 18 Kd
Figure 3: Protein Induction and Purification of Hel-
N l with the Biotinylatable Tag at the C-terminal.
Protein induction can be seen in the t=0, t=3 and SS lanes.
The lanes containing t=0 and t=3 are time before induction of
IPTG and the time 3 hours after induction of IPTG respectively.
Sonication of the cells which resulted in releasing all the protein
into the supernatant can be seen in lane SS. The protein was
purified with increasing concentrations of imidizole ranging from
S mM to 250 mM as seen in the figure. The migration of the
Hel-Nl protein is approximately 40 kD as expected. As the
concentration of imidizole increases the band becomes sharper
and more distinct. The optimum concentration and purity for the
protein is seen with the 150 mM elution buffer.
5 2 2 2 2
2 1 s S s g
E © © ® ®
«L CO un — — N (N
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
to be approximately 40 kD. The protein was induced and eluted from the nickel beads at
elution buffers 50 to 200 mM of imidazole. As the concentration of imidazole in the
elution buffers increases, the band migrating at approximately 40 kD appears to be
sharper and more distinct. The optimum concentration and purity for the protein is seen
with the 150 mM elution buffer. The concentration of the purified protein was estimated
to be 1 ug/10 ul or 100ng/ ul. Protein production was also induced using the Hel-Nl
vector containing the biotinylatable tag located at the N-terminus. Figure 4 shows Hel-N 1
to be migrating at well above the 42kD manner, which is higher than expected. The
biotin on the Hel-Nl may be responsible for the slower migration of the protein.
Protein and biotinvlation determination
To determine if the protein was biotinylated, a western blot was generated and probed
with extravidin horseradish peroxidase (HRPO), which will bind to biotinylated protein
and allow visualization of the protein through chemiluminescent detection. The Hel-Nl
protein that contained the C-terminal biotinylatable tag was assayed by western blot
analysis first. The blot that contained the Hel-Nl with the C-terminal tag was exposed to
film for 1 second, 30 seconds, 1 minute and overnight. The overnight exposure, shown in
figure 5 shows there is very little biotinylation of the Hel-Nl protein. A very faint band
can be seen in the Hel-Nl initial lane, this is the amount of biotinylated protein added
prior to avidin bead incubation. The Hel-Nl supernatant band also has a faint band
representing the inability of biotinylated protein to bind to the avidin beads.
22
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
w
u
«
s
I.
-C
©
u
©
"1
S
IT )
SI
s
©
IT )
a a a a
e s s e
© © © ©
© i/s © in
— — < fS fN
80 kD -
42 kD -
32 Id) —
18 k D -
Figure 4: Protein Induction and Purification of Hel-Nl with
the Biotinylatable Tag at the N-terminus.
Protein induction can be seen in lanes t=0 and t=3, which
represent time before induction and time three hours after
induction. The protein was purified with increasing concentrations
of imidizole ranging from 5 mM to 250 mM. The most optimum
concentration and purity of the protein can be seen in the 250 mM
elution buffer lane. The migration of Hel-Nl which appears to be
significantly above 42 kD, is higher than expected. The biotin on
the Hel-Nl may be responsible for the slower migration of the
protein.
23
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
244 Kd —
42 Kd —
32 Kd —
18 Kd —
Figure 5: Low Amounts of Biotinylatation with the C-terminal
Biotinylatable tag.
A western blot was probed with extravidin horse radish peroxidase
(HRPO) and exposed overnight. There is very little signal present in the lanes
containing the Hel-Nl initial, and Hel-Nl sup of the avidin bead binding assay.
The initial amount of protein that was added to the avidin beads are represented in
the Hel-Nl initial lane. The Hel-Nl supernatant lane represents the amount of
Hel-Nl protein that was not able to bind to the avidin beads and can be
considered as non-biotinylated. However, there is signal in the Hel-N 1 sup lane
which may signify the protein is biotinylated but is unable to bind to the beads.
This can be compared to the Hel-Nl boiled lane where there is no signal present.
The protein may not be biotinylated or unable to bind to the beads. The control
lanes (MY initial, MY supernatant, MY boiled) were non biotinylated proteins
and were expected not to give any signals. There are two prominent bands seen at
approximately 32 kD. These bands can be the degraded products resulting from
the Hel-Nl protein. The biotinylatable tag may have been degraded which can
account for the low amount of protein biotinylated.
24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
When the beads were boiled to release biotinylated protein bound to beads, there
was no signal present (Hel-Nl boiled lane). The more prominent bands seen migrating
approximately at 35 kD are the degradation products of the Hel-Nl protein. The
biotinylated tag may have been degraded thus resulting in the low amount of biotinylated
protein. This amount of biotinylation is not sufficient to bind enough Hel-Nl to avidin
coated BIAcore chips. I concluded form these results that protein can not be efficiently
biotinylated from a biotinylatable tag located at the C-terminus.
The protein with the biotinylatable tag at the N terminus was assayed next. After
the addition of the extravidin HRPO, the film was exposed for 1 second, 2 seconds, 3
seconds, and 5 seconds. Fig 6 shows that a very strong signal was obtained, indicating a
high level of biotinylation. However, where the protein is expected to migrate, the signal
burned a transparent area within the all the dark overexposed film. I concluded from the
results that the protein containing the biotinylatable tag at the N-terminal can be
biotinylated more successfully and efficiently than the protein with the C-terminal tag.
Amount of protein biotinylated
Since I had shown that the N-terminal biotinylatable tag appeared to be
functional, the next step was to determine the efficiency of biotinylation. An assay
involving streptavidin paramagnetic beads acquired from Dynal was used to determine
the amount of protein that was biotinylated. I compared the concentrations of protein
initially added to the beads to the concentrations of the protein that could not bind to the
beads. Since the streptavidin beads have a strong binding affinity for biotin, I expect all
25
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
L.
Q J
J C
_ 2 2 2 2
s | E E E E
* j f ” E o o S o §
J L 4 L u ) M t > t > c m n
244 Kd—
80 Kd—
42 K d -
18 K d -
Figure 6: Successful Biotinylatation of Hel-Nl
Containing the Biotinylatable Tag at the N-terminus.
A western blot was probed with extravadin horse
radish peroxidase (HRPO), when exposed for 1 second it
exhibited a strong signal indicating a high level of
biotinylation. The strength of the signal caused the film to
become overexposed and where the expected protein was to
migrate (above 40 kD) was left a burned transparent area
within the dark field of the film.
26
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
the protein that was biotinylated to bind to the beads while all unbiotinylated protein
would remain in the supernatant.
The supernatant (unbound) represents all the protein that was not biotinylated and
could not bind to the beads. The lanes containing the dilutions of the eluted bound
protein were compared to the supernatant containing lane to determine which dilution is
most comparable to the unbound protein. Figure 7 shows that the most diluted protein
sample, the 32-fold dilution, still contains slightly more protein than the supernatant
(unbound). Thus, I conclude that for every 1 unbiotinylated protein, there are at least 32
biotinylated proteins, indicating that over 97% of the Hel-Nl purified protein was
biotinylated.
Ability of protein to bind antibody in native form
In order to determine whether the addition of a single biotin influences the ability
of the antibodies to bind to the native protein, an immunoprecipitation assay was
performed. A possible concern was that the biotin might interfere with the epitope on the
protein that is recognized by the anti-Hu antibodies. Sodeyama described that the epitope
on the Hu proteins that is required for antibody binding is located within RRM1 and
RRM2 (53). This is located near the N-terminus of the gene and might be hindered by
the biotin. Following an incubation of biotinylated Hel-Nl with the rabbit anti-HuD
antibody, protein G beads were used to precipitate the antibodies. The precipitate and
several controls were analyzed by western blot analysis (fig.8). The blot was probed with
the indicated antibodies and alkaline phosphatase was used to obtain signal.
27
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 7: Determining the Amount of Protein Biotinylated
The efficiency of biotinylatation was determined for the N-terminal
biotinylatable tag by a bead binding assay and analyzed by western blot. The
initial input lane represents the amount of protein that was added before binding
to the avidin beads. The amount of protein that is not biotinylated is in the
unbound lane. The biotinylated protein was bound to avidin beads, boiled and
serially diluted 2 fold. There is signal present in all the lanes indicating
biotinlyation of the protein. The most diluted sample (32-fold dilution) still
contains slightly more protein than the unbound lane. For every 1 unbiotinylated
protein, there are at least 32 biotinylated proteins which yields a 97%
biotinylation efficiency.
28
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
V C V) N
4» B > V U
S e S s c e
S B « S B s b S S s s
mi -J mi -J -J mi
80 kD ~
C M
**
I
Q
32 kD -
18
I
Q
.X
7kD -
Protein
Hel-N1 B-Hel BSA Hel-N1 Hel-N1 B-Hel
Antibody G-anti M — anti-Hu anti- anti-Hu anti-Hu
biotin Myc
Figure 8: Ability of Hel-Nl to be Bound by Anti-Hu
antibodies
To determine if an addition of a single biotin can influence the
ability of antibodies to bind to native protein, an immuno precipitation
assay was performed and analyzed on western blot. The signal in lane 1
indicates that biotinylated Hel-N 1 protein can be bound by anti-Hu
antibodies. Unbiotinylated Hel-N 1 can also bind anti-Hu antibodies and
anti-Myc antibodies indicating the reliability of the procedure (lanes 2 and
3). To exclude background and non specific binding, lane 4 represents
albumin’s inability to bind anti-Hu antibodies. Lane 5 represents the
possibility that the signal seen in lane I was due to residual unbound
protein not removed during washing. The overexposure produced in lane
6 is due to the secondary antibody (conjugated to a biotin) that cross
reacted with the reagent used to probe the filter (HRPO). The higher
bands present are due to the presence of the antibody heavy chain. The
addition of a biotin does not affect the ability of anti-Hu antibodies to
bind to biotinylated Hel-Nl.
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Lane 1 shows a strong signal at the position where the Hel-Nl protein is expected
to migrate slightly higher than the 42 kD marker indicating the presence of protein. This
signal indicates that the anti-HuD antibodies was able to bind the biotinylated Hel-Nl
protein without interference due to the addition of biotin. The HEL-N 1 protein with no
biotinylation was also tested for anti-Hu antibody binding. For comparison,
unbiotinylated Hel-Nl protein was immunoprecipitated with the rabbit anti-HuD
antibody (Lane 2). This indicates the presence of protein and the ability of the mouse
anti-HuD antibody to bind to Hel-Nl. Lane 3 shows the ability of unbiotinylated Hel-Nl
to be immunoprecipitated with a mouse anti-Myc antibody. There is a Myc epitope tag
located at the C-terminus of Hel-Nl. The binding of the mouse anti-Myc antibody to the
Myc epitope indicates that antibodies can be immunoprecipitated out by this procedure.
In order to distinguish between the signal in lane 1 and non-specific binding, albumin and
anti-HuD antibody was run in lane 4. Albumin did not bind anti-HuD antibodies and
there is no protein present. Since albumin does not contain the epitope required for anti-
HuD binding, there is no signal seen in the 42 kD range. To exclude the possibility that
the signal in lane 1 was derived form residual unbound protein that was not removed
during washing, unbiotinylated protein without antibody was incubated in parallel (lane
5). There was no signal present in the 42 kD range where the Hel-Nl protein is expected.
Lane 6 contains the Hel-Nl protein with a secondary antibody that is conjugated with a
biotin. This biotin causes the overexposure due to the cross reactivity with the reagent
used to probe the filter (extravidin HRPO). The higher bands seen are due to the
presence of the antibody heavy chain. I conclude from these results that the addition of a
biotin does not affect the ability of the protein to be bound by anti-Hu antibodies.
30
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Discussion
Improving our means for early detection and diagnosis of lung cancer is
extremely important and can be a useful tool in the battle towards increasing the survival
of lung cancer patients. Hu proteins, which for unknown reasons are found in all SCLC,
give rise to an immune response in at least 16% of patients, thus could be a useful in the
early detection of SCLC. Why do these Hu proteins elicit an immune response in a
subset of patients? One possibility may be that they are mutated, and that the body is
responding to the mutated (and therefore foreign) protein. Hu proteins may be necessary
to terminate the neuroblast cell cycle and promote neuronal differentiation and maintain
the neuronal phenotype. A mutation in the Hu protein may lead to a loss of suppressor
function and recommencement of the cell cycle. Thus mutated Hu proteins produced in
SCLC may be involved in uncontrolled cell cycling. This unregulated cell proliferation
and cell cycling could result in malignant transformation and tumorigenesis. The
resulting tumor could continue to produce mutant Hu proteins which might be recognized
by the immune system as foreign. These foreign Hu proteins would lead to the
production of anti-Hu antibodies by the immune system and could thereby lead to
PEM/PSN.
However, searches for mutations in the genes form SCLC cells have as yet
yielded no evidence for mutations (16). A second possibility is that Hu genes may
somehow be activated and expressed at high levels without mutations in the gene. The
immune response may then be due to the expression of neuronal proteins that are not
normally expressed in the lung. A question that arises when studying anti-Hu antibodies
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
is why some patients exhibit a low or high immunologic response to the Hu antigen
expressed in the tumor. A theory presented by Dalmau, was that the neuron specific Hu
proteins are not presented to the immune system during the establishment of immune
tolerance (12). Graus, shown that MHC proteins are not expressed in neurons (25). The
de novo expression of the Hu proteins in tumors that do express the MHC would then
result in a profound immune reaction (12). The magnitude of the immune response
would dictate the titer of the anti-Hu antibodies.
Whatever the origin and mechanism of the anti-Hu response, the ability to detect
anti-Hu antibodies in patient sera might aid in the survival rate of lung cancer. With the
use of BIAcore, a cloned Hu protein can be coupled to a sensor chip by biotin avidin
interactions and detect anti-Hu antibodies in SCLC patient sera. In separate ways, the
Hel-Nl protein was attempted to be biotinylated by inserting a biotinylatable tag at the N-
terminus and C-terminus of the Hel-N 1 gene. By cloning a biotinylatable tag into the
Hel-Nl vector, a biotin ligase (BirA) can biotinylate the Hel-Nl protein at the specified
tag in vivo, inside the E.coli host. A Hel-Nl protein carrying a C-terminal biotinylatable
tag showed very little biotinylation. Several factors could account for this. The
biotinylatable tag may not have been accessible due to steric hindrance. However the C-
terminal tag is located next to the His tag which is used in binding nickel beads for
protein purification. The ability of Hel-Nl to be purified by the His tags contradicts the
theory of steric hindrance. Another possible explanation may be the tag was not optimal
for this context. There could also be a mutation within that tag rendering it useless for
recognition. This was refuted when the tag was sequenced and the results showed no
mutations present. A possible explanation that is the most reasonable is the partial
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
degradation of the protein at the biotinylatable tag (figure 4). When protein induction
and biotinylation was performed in-vivo, using a Hel-Nl protein carrying a N-terminal
biotinylatable tag, this yielded a biotinylated Hel-Nl protein. A dilution experiment
showed that over 97% o f Hel-Nl were biotinylated. This made it possible to attempt a
trial run using BIAcore to bind anti-Hu antibodies from sera.
The biotinylated Hel-N 1 was added and bound to a streptavidin coated BIAcore
chip. High concentrations of the biotinylated Hel-Nl were easily bound on to the sensor
chip. Several dilutions of sera including rabbit anti-Hu and human anti-Hu serum form a
SCLC/PEM patient were passed over the chip. Results showed little to no binding of
anti-Hu antibodies to the Hel-Nl coated BIAcore chip. (Data not shown) There are
several possibilities that may have attributed to the lack of binding of anti-Hu antibodies
to the Hel-Nl surface. The BIAcore chips are extremely expensive and can be coated
only once. However, thereafter the surface can be used for many assays to bind to the
coated chip. For my first preliminary trial it was decided to attempt the assay using an
old chip which might account for the failed attempt. The chip may have not been
functioning optimally due to its age. However, efficient binding of the biotinylated Hel-
Nl to the sensor chip indicated that the chip was still functional. Thus there may be other
possibilities for the lack of antibody binding.
The rabbit sera used was not purified and could contain many contaminants. In
addition, the rabbit anti-Hu antibodies were generated in rabbits that had been immunized
with a fragment of HuD containing RRM1 and RRM2. The choice of HuD RRM &
RRM2 to immunize the rabbits was based on the fact that anti-Hu antibodies should be
able to cross react with all the Hu proteins and the main epitopes are located in RRM1
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and RRM2 (53). The production of large quantities of the protein required for
immunization was easier for the RRM1 & RRM2 fragment than the production of full
length Hu proteins. It is possible that the antibodies produced in response to truncated
HuD proteins differ than from those produced against full length proteins. This does not
seem likely since the antibodies do react on western blot with all four of the Hu proteins
(data not shown). The same problems did not apply to the human serum as the rabbit
serum, however, the human serum is extremely precious so a limited amount was used as
a preliminary test.
Past studies have shown that Hu proteins can be bound by antibodies under the
denaturing conditions of western blot analysis (11). It may be possible that the native
form of the proteins used in BIAcore can not bind anti-Hu antibodies as well. Denaturing
conditions could open up the structure allowing for a different manner of antibody
binding. In addition the biotinylation of Hel-Nl could also interfere with the epitope
recognized by the antibody. These possibilities were excluded by an
immunoprecipitation assay. (Fig 8) The results showed that the biotinylated Hel-Nl
protein in native form can be bound by anti-Hu antibodies just as well as unbiotinylated
proteins. Although the experiment showed that antibodies were able to bind free
biotinylated Hel-Nl protein, the ability to bind biotinylated Hel-Nl protein that is
coupled to the BIAcore sensor chip may be hampered. The binding of the biotinylated
Hel-Nl to the chip could cause the protein to be pulled in a certain direction and might
change its confirmation. Such an alteration in protein structure might then not allow the
antibodies to bind to the protein. Several possibilities that may eventually lead to success
would be the use of a new chip, purified rabbit antibodies, or a different fragment of Hu.
34
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
I have successfully cloned a biotinylatable tag in to the N-terminus of the Hel-Nl
vector. The in vivo biotinylation within the E.coli host was shown to be 97% effective
and reproducible method to adding a single biotin onto a cloned Hu protein. This method
which is not yet used widely, will be very useful not only for BIAcore experiments but
for a variety of applications in the lab. I have shown that the antibodies raised against
HuD RRM1&2 are cross reactive with the Hel-Nl protein. The ability of the antibody to
bind to the protein is not hindered by the addition of the biotin. BIAcore can be a
powerful tool for early detection of lung cancer due to its sensitivity, its ability to identify
binding in real time and the speed of the assay. This method does not require lengthy
washing such as in western blot analysis where the loss of antibodies during each wash
may occur. If the method is successful, then it could also be applied to the detection of
other cancers that have associated immune response such as breast cancer, Hodgkin’s
lymphoma or colon.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Bibliography
1. Ahrendt S, Chow J, Xu L, Yang S, Eisenberger C, Esteller M, Herman J, Wu L,
Decker A, Jen J, Sidransky D. Molecular Detection of Tumor Cells in
Bronchoalveolar Lavage Fluid from Patients with Early Stage Lung Cancer. Journal
of the National Cancer Institute 1999,91:332-339
2. Anderson N, Rosenblum M, Chaus F, Wiley R, Posner J. Autoantibodies in
Paraneoplastic Syndromes Associated with Small Cell Lung Cancer. Neurology
1988,38:1391-1398
3. Antoine J, Mosnier J, Honnorat J, Convers P, Absi L, Lapras J, Michel D.
Paraneoplastic Demyelinating Neuropathy, Subacute Sensory Neuropathy, and Anti-
Hu Antibodies: Clinicopathological Study of An Autopsy Case. Muscle and Nerve
1998,21:850-857
4. Ball N, King P Neuron-specific Hel-Nl and HuD as Novel Molecular Markers of
Neuroblastoma: A Correlation of HuD Messenger RNA Levels with Favorable
Prognostic Features. Clinical Cancer Research 1997, 3:1859-1865
5. Bayer E, Wilchek M. Biotin-Binding Proteins: Overview and Prospects. Methods in
Enzymology 1990, 184:49-52
6. Byrne T, Mason W, Posner J, Dalmau J. Spontaneous Neurological Improvement in
Anti-Hu Associated Encephalomyelitis. Neurol Neurosurg Psychiatry 1997,62:276-
278
7. Cairns P, Okami K, King P, Bonacum J, Ahrendt S, Wu L, Mao L, Jen J, Sidransky
D. Genomic Organization and Mutation Analysis of Hel-Nl in Lung Cancer with
Chromosome 9P21 Deletions. Cancer Research 1997, 37:5356-5359
8. Carney D. Lung Cancer Hodder Headline Group, 1995 pp3-l91
9. Cho J, Noguchi M. Expression of HuD (A Paraneoplastic Encephalomyelitis
Antigen) mRNA in Lung Cancer. JKMS 1997,12:305-310
10. Dalmau J, Fumeaux H, Rosenblum M, Graus F, Posner J. Detection of the Anti-Hu
Antibody in Specific Regions of the Nervous System and Tumor from Patients with
Paraneoplastic Encephalomyelitis/Sensory Neuronopathy. Neurology 1991,41:1757-
1764
11. Dalmau J, Fumeauz M, Gralla R, Kris M, Posner J. Detection of the Anti-Hu
Antibody in the Serum of Patients with Small Cell Lung Cancer- A Quantitative
Western Blot Analysis. Ann Neurol 1990,27:544-552
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12. Dalmau J, Fumeaux C, Posner J. The Expression of the Hu (Paraneoplastic
Encephalomyelitis/Sensory Neuronopathy) Antigen in Human Normal and Tumor
Tissues. Am J Pathol 1992, 141:881-886
13. Dalmau J, Graus F, Cheung N, Rosenblum M, Ho A, Canete A, Thompson S,
Posner J. Major Histocompatibility Proteins, Anti-hu Antibodies, and Paraneoplastic
Encephalomyelitis in Neuroblastoma and Small Cell Lung Cancer. Cancer 1995,
75:99-109
14. Dalmau J, Graus F, Rosenblum M, Posner J. Anti-Hu-Associated Paraneoplastic
Encephalomyelitis/Sensory Neuronopathy. Medicine 1992, 71:59-72
15. Dalmau J, Posner J. Neurologic Paraneoplastic Antibodies. Neurology 1994,
44:2241-2246
16. Dropcho E. Priciples of Paraneoplastic Syndromes. Annals New York Academy of
Sciences 1998, 841:246-261
17. Dropcho E, King P. Autoantibodies Against the Hel-Nl RNA-binding Protein
Among Patients with Lung Carcinoma: An Association with Type I Anti-Neuronal
Nuclear Antibodies. Annals of Neurology 1994,36:200-205
18. Fan C, Steitz J. HNS, a Nuclear-cytoplasmic Shuttling Sequence in HuR. Proc. Natl.
Acad. Sci 1998, 98:15293-15298
19. Fan X, Steitz J. Overexpression of HuR, a Nuclear-Cytoplasmic Shuttling Protein,
Increases the in vivo Stability of ARE-containing mRNAs. The Embo Journal 1998,
19:3448-3460
20. Gao F, Carson C, Levine T, Keene J. selection of a Subset of mRNAs from
Combinatorial 3’Untranslated Region Libraries Using Neuronal RNA-binding Protein
Hel-Nl
21. Gao F, Keene J. Hel-Nl/Hel-N2 Proteins are Bound to Poly (A)+ m RNA in
Granular RNP Structures and are Implicated in Neuronal Differentiation. Journal of
Cell Science 1996,109:579-589
22. Gazdar A, Minna J. Molecular Detection of Early Lung Cancer. Journal of the
National Cancer Institute 1999, 91:299-301
23. Good P. A Conserved Family of ELAV-like Genes in Vertebrates. Proc. Natl. Acad.
Sci. 1995,92:4557-4561
24. Graus F, Riballa T, Campo E. Immunohistochemical Analysis of the Immune System
in Paraneoplastic Encephalomyelitis. Neurology 1990,40:219-222
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
25. Graus F, Tora R, Malats N, Verschuuren J, Cardenal F, Vinolas N, del Muro G,
Vadell C, Mason R, Posner J, Real F. Anti-hu Antibodies in Patients with Small Cell
Lung Cancer: Association with Complete Response to Therapy and Improved
Survival. Journal of Clinical Oncology 1997, 15:2866-2872
26. Graus Y, Verschuuren J, Degenhardt A, van Breda Vriesman P, Baets M, Posner J
Burton D, Dalmau J. Selection of Recombinant Anti-HuD Fragments from a Phage
Display Antibody Library of a Lung Cancer Patient with Paraneoplastic
Encephalomyelitis. Journal of Neuroimmunology 1998, 82:200-209
27. Greenlee J, Parks T, Jaeckle A. Type Iia (anti-Hu) Antineuronal Antibodies Produce
Destruction of Rat Cerebellar Granule Neurons in vitro. Neurology 1993,43:2049-
2054
28. Han J, Knops J, Longshore J, King P. Localization of Human ELAV-like Neuronal
Protein 1 (Hel-Nl) on Chromosome 9p21 by Chromosome Microdissection
Polymerase Chain Reaction and Flourescence in Situ Hybridization. Genomics 1996,
36:189-191
29. Jain R, Andrews L, McGowan M, Pekala P, Keene J. Ectopic Expression of Hel-Nl,
an RNA-binding Protein, Increases Glucose Transporter (GLUT1) Expression in 3T3
Adipocytes. Molecular and Cellular Biology 1997, 17:954-962
30. King P. Cloning the 5’ Flanking Region of Neuron-Specific Hel-Nl: Evidence for
Positive regulatory Elements Governing Cell-Specific Transcription. Brain Research
1995,723:141-147
31. King P, Dropcho E. Expression of Hel-Nl and Hel-N2 in Small Cell Lung
Carcinoma. Ann Neurol 1996, 39:679-681
32. King P, Levine T, Fremeau R, Keene J. Mammalian Homologs of Drosophila ELAV
Localized to a Neuronal Subset Can Bind in vitro to the 3; UTR of mRNA Encoding
the Id Transcriptional Repressor. Journal of Neuroscience 1994,14:1943-1952
33. Kostyk S, Wheeler E, Wagner J. Unusual Expression of the Hu Paraneoplastic
Antigen in the Visual System. Neuro Report 1996,7:1549-1552
34. Lafon L, Carballes F, Brewer G, Poiret M, Morello D. Developmental Expression of
AUF1 and HuR, Two c-myc mRNA Binding Proteins. Oncogene 1998, 16:3413-
3421
35. Levine T, Gao F, King P, Andrews L, Keene J. Hel-Nl: an Autoimmune RNA-
Binding Protein with Specificity for 3’ Uridylate-Rich Untranslated Regions of
Growth Factor mRNAs. Molecular and Cellular Biology 1993, 13:3494-3504
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
36. Liu J, Dalmau J, Szabo A, Rosenfeld M, Huber J, Fumeaux H. Paraneoplastic
Encephalomyelitis Antigens Bind to the AU-Rich Elements of mRNA. Neurology
1995, 45:544-550
37. London S, Daly A, Cooper J, Navidi W, Carpenter C, Idle J. Polymorphism of
Glutathione S-Transferase Ml Lung Cancer Risk Among African-Americans and
Caucasians in Los Angeles County, California. Journal of the National Cancer
Institute 1995, 87:1246-1253
38. Ma W, Cheng S, Campbells C, Wright A, Fumeaux H. Cloning and Characterization
of HuR, a Ubiquitously Expressed Elav-like Protein. Journal of Biological Chemistry
1996, 271:8144-8151
39. Ma W, Chung S, Fumeaux H. The Elav-like Proteins Bind to AU-rich Elements and
to the Poly (A) Tail of mRNA. Nucleic Acids Research 1997,25:3564-3569
40. Manley G, Smitt P, Dalmau J, Posner J. Hu Antigens: Reactvity with Hu Antibodies,
Tumor Expression, and Major Immunogenic Sites. Annals of Neurology 1995,
38:102-110
41. Marusich M, Fumeaux H, Henion P, Weston J: Hu neuronal Proteins are Expressed
in Proliferating Neurogenic Cells. Journal of Neurobiology 1993,25:143-155
42. Merril J. Autoimmune Disease and the Nervous System. Western Journal of
Medicine 1992, 156:639-646
43. Okano H, Darnell R. a Hierarchy of Hu RNA Binding Proteins in Developing and
Adult Neurons. Journal of Neuroscience 1997, 17:3024-3037
44. Peng S, Chen C, Xu N, Shyu A. RNA Stabilization by the AU-rich Element Binding
Protein, HuR, and ELAV protein. The Embo Journal 1998, 17:3461-3470
45. Pharmacia BIAtechnology Handbook.(Pharmacia Biosensor AB)1994
46. Posner J. the Anti-Hu Syndrome: A Model Paraneoplastic Disorder. Clinical Neuro
oncology
47. Posner J, Dalmau J. Paraneoplastic Syndromes. Current Opinion in Immunology
1997, 9:723-729
48. Sakai K, Ogasawasa G. Analysis of Autoantibody Binding to 52 kD Paraneoplastic
Cerebellar Degeneration- Associated Antigen Expressed in Recombinant Proteins.
Ann, Neurol 1993,33:373-380
49. Schottenfeld D, Epidemiology of Lung Cancer(Lung Cancer: Principles and Practice
ed), pp305-321Philadelphia:Raven
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
50. Sellers T, Potter J, Bailey J, Rich S, Rothschild H, Elston R. Lung Cancer Detection
and Prevention: Evidence for an Interaction Between Smoking and Genetic
Predisposition. Cancer Research 1992, 52:2694-2697
51. Smitt P, Manley G, Dalmau J, Posner J. The HuD Paraneoplastic Protein Shares
Immunogenic Regions Between PEM/PSN and Several Strains and Species of
Experimental Animals. Journal of Neuroimmunology 1996, 71199-206
52. Smitt S, Manley G, Posner J. Immunization with the Paraneoplastic
Encephalomyelitis Antigen HuD does not Cause Neurologic Disease in Mice.
Neurology 1995,45:1873-1878
53. Sodeyama N, Ishida K, Jaeckle K, Zhang L, Azuma A, Yamada M, Mizusawa H,
Wada Y. Pattern of Epitopic Reactivity of the Anti-Hu Antibody on HuD With and
Without Paraneoplastic Syndrome. Neurol Neurosurg Psychiatry 1999,66:97-99
54. Steller U, Kohls S, Muller B, Soller R, Muller R, Schlender J, Blohm D. The RNA
Binding Protein HuD: Rat cDNA and Analysis of the Alternative Spliced mRNA in
Neuronal Differentiating Cell Lines P19 and PC 12. Molecular Brain Research 1995,
35:285-296
55. Szabo A, Dalmau J, Manley G, Rosenfeld M, Wong E, Henson J, Posner J, Fumeaux
H. HuD, a Paraneoplastic Encephalomyelitis Antigen, Contains RNA-Binding
Domains and is Homologous to Elav and Sex-lethal. Cell 1991, 67:325-333
56. Tora M, Graus F, de bolos C, Real F. Cell Surface Expression of Paraneoplastic
Encephalomyelitis/Sensory Neuronopathy Associated Hu Antigens in Small Cell
Lung Cancers and Neuroblastomas. Neurology 1997,48:735-741
57. Travis W, Linder J, Mackay B. Classification, Histology, Cytology, and Electron
Microscopy (Lung Cancer: Principles and Practice ed), pp361-395Philadelphia:Raven
58. Wilchek M, Bayer E. Introduction to Avidin-Biotin Technology. Methods in
Enzymology 1990,184:5-11
59. Wolpaw D: Early Detection in Lung Cancer. Medical Clinics of North America
1996, 80:63-81
60. Wong R, Mytych D, jacobs S, Bordens R, Swanson S. Validation Parameters for a
Novel Biosnesor Assay Which Simultaneously Measures Serum Concentrations of a
Humanized Monoclonal Antibody and Detects Induced Antibodies. J Immunol 1997,
10:1-15
40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Dual functions of Vav in Ras-related small GTPases signaling regulation
PDF
A transgenic mouse model for small cell lung cancer
PDF
Development of lung cancer specific DNA methylation markers
PDF
Analysis of the HSD3B2 gene in prostate cancer
PDF
Development and secretions of salivary glands using mouse models
PDF
A TNF alpha-responsive kinase activity may play a key role in IKK activation
PDF
Expression of matrix metalloproteinases and their inhibitors in the muscles of amyotrophic lateral sclerosis and control patients
PDF
Biochemical analysis of somatic mutations in steroid 5alpha-reductase type II in prostate cancer
PDF
Association between single nucleotide polymorphisms in the 3'untranslated region of the SRD5A2 gene and prostate cancer risk
PDF
Characterization of target cell entry by murine leukemia viruses
PDF
A review of molecular conjugates and their use in gene therapy with the presentation of a model experiment: Gene therapy with novel fusion proteins that target breast cancer cells
PDF
Colorectal cancer risks in Singapore Chinese: Polymorphisms in the insulin-like growth factor-1 and the vitamin D receptor
PDF
Investigation of the role of epigenetic modification of DNA and chromatin in aberrant gene silencing in cancer cells
PDF
Identification of DNA methylation markers for lung cancer and mesothelioma
PDF
Transcriptional targeting of retroviral vector replication to prostate tumor cells
PDF
Lung cancer, myc gene deregulation and Hu gene expression
PDF
A study of pediatric oncology nurses' attitudes to and knowledge of genetic testing
PDF
Study of matrix metalloproteinases in hepatic veno-occlusive disease
PDF
Establishment and properties of a stable transfected epicardial cell line expressing a dominant negative retinoic acid receptor
PDF
Using the grp78 and VEGF promoter as components for specific expression in a tumor environment and the effect of genistein treatment on grp78 induction in relation to breast cancer cells
Asset Metadata
Creator
Tsou, Jeffrey An-Pang
(author)
Core Title
Detection of anti-Hu antibodies, a possible key to early diagnosis of small cell lung cancer
School
Graduate School
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
biology, molecular,health sciences, oncology,OAI-PMH Harvest
Language
English
Contributor
Digitized by ProQuest
(provenance)
Advisor
[illegible] (
committee chair
), [illegible] (
committee member
), Zandi, Ebrahim (
committee member
)
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c16-32414
Unique identifier
UC11336759
Identifier
1397649.pdf (filename),usctheses-c16-32414 (legacy record id)
Legacy Identifier
1397649.pdf
Dmrecord
32414
Document Type
Thesis
Rights
Tsou, Jeffrey An-Pang
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA
Tags
biology, molecular
health sciences, oncology