Molecular Biochemistry III
(1702805)
By
Samah El-Sayed Diab
Application of Northern
Blotting Technique in Cancer
Disease
6/18/2020
Applied Medical Chemistry Department
CONTENTS
Introduction to Cancer Disease
The Hallmarks of Cancer
Cancer Risk Factors
Types of Cancer
Pathogenesis of Cancer
Diagnosis of Cancer & Northern Blotting Technique
Treatment of Cancer
References
1) Introduction
Cancer is a group of diseases characterized by uncontrolled cell
growth and proliferation whereby cells have escaped the body’s normal
growth control mechanisms and have gained the ability to divide
indefinitely. It is a multi-step process that requires the accumulation of
many genetic changes over time (Figure 1). These genetic alterations
involve activation of proto-oncogenes to oncogenes, deregulation of
tumour suppressor genes and DNA repair genes and immortalization. (1)
Figure 1: Overview of the road to cancer
Cancer
II) The Hallmarks of Cancer (2)
Growth signal autonomy
Evasion of growth inhibitory signals
Unlimited replicative potential
Invasion and metastasis
Angiogenesis (formation of new blood vessels)
Evasion of cell death
Genome instability and mutation
Avoiding immune destruction
Tumor-promoting inflammation
Reprogramming energy metabolism
III) Cancer Risk Factors
It is not possible to find out the specific cause for cancer. Cancer
cells are modulated by culture condition and extracellular
microenvironment condition. (3)
But there are many risks which increase
the cancer such as:
Age (4,5)
Heredity (6)
Radiation (7)
Reproduction and hormones (8)
Dietary factors (9)(10)
Obesity (11)
Alcohol (12)
Smoking (13)
Infections (14)(15)
Occupational exposures, e.g. many chemicals,
radioactive materials and asbestos (16)
IV) Types of Cancer
Cancers are classified by the type of cells that constitutes the tumor
and, therefore, the tissue presumed to be the origin of the tumor into: (17)
A. Carcinoma: cancer that affects the epithelial tissues that lines
internal organs. The most common cancers like breast, prostate,
lung and colon cancer come under this category.
B. Sarcoma: cancer that begins in connective or supportive tissue
(e.g., bone, cartilage, fat, muscle, blood vessels).
C. Leukemia: cancer related to blood-forming tissue.
D. Lymphoma: cancers that affects the lymphatic tissue.
E. Myeloma: cancer that begins in bone marrow.
F. Blastoma: cancer that begins in embryonic tissue.
G. Central nervous system cancers: cancers that begin in the tissues
of the brain and spinal cord.
V) Pathogenesis of Cancer
Carcinogenesis is a multistage process. The application of a cancer-
producing agent (carcinogen) does not lead to the immediate production
of a tumor. There are a series of changes after the initiation step induced
by the carcinogen (Figure 2).
1- Initiation
It is essentially irreversible changes in appropriate target somatic
cells. In the simplest terms, initiation involves one or more stable cellular
changes arising spontaneously or induced by exposure to a carcinogen.
This is considered to be the first step in carcinogenesis, where the cellular
genome undergoes mutations, creating the potential for neoplastic
development, which predisposes the affected cell and its progeny to
subsequent neoplastic transformation. (18, 19)
2- Promotion
Once a cell has been mutated by an initiator, it is susceptible to the
effects of promoters. These compounds promote the proliferation of the
cell, giving rise to a large number of daughter cells containing the
mutation created by the initiator. Promoters have no effect when the
organism in question has not been previously exposed to an initiator.
Unlike initiators, promoters do not covalently bind to DNA or
macromolecules within the cell. Many bind to receptors on the cell
surface in order to affect intracellular pathways that lead to increased cell
proliferation. Promoters are often specific for a particular tissue or
species due to their interaction with receptors that are present in different
amounts in different tissue types. (18)
3- Progression
It is the process through which successive changes in the neoplasm
give rise to increasingly malignant sub-populations. Molecular
mechanisms of tumor progression are not fully understood, but mutations
and chromosomal aberrations are thought to be involved. The process
may be accelerated by repeated exposures to carcinogenic stimuli or by
selection pressures favoring the autonomous clonal derivatives. The
initiated cells proliferate causing a fast increase in the tumor size. As the
tumor grows in size, the cells may undergo further mutations, leading to
increasing heterogeneity of the cell population. (20)
Figure 2: Pathogenesis of Cancer
VI) Diagnosis of Cancer
1) Physical Exam
The physician may feel areas of patient's body for lumps that may
indicate a tumor. During a physical exam, also the physician may look for
abnormalities, such as changes in skin color or enlargement of an organ
that may indicate the presence of cancer. (21)
2) Biomarkers
Biomarkers are proteins which are released from cancers and whose
detection or increase in the serum may screen or confirm the presence of
certain cancers. Biochemical assays for tumor-associated enzymes,
hormones and other markers are not being used for the definitive
diagnosis of cancer. Instead, cancer biomarkers play a role in the early
detection, outcome prediction and detection of disease recurrence. (22)
3) Imaging Tests
Imaging tests allow examination of bones and internal organs in a
noninvasive way. Imaging tests used in diagnosing cancer may include
a computerized tomography (CT) scan, bone scan, magnetic resonance
imaging (MRI), positron emission tomography (PET) scan, ultrasound
and X-ray. (23)
4) Biopsy
During a biopsy, a sample of cells is collected for testing in the
laboratory. There are several ways of collecting a sample and this
depends on type of cancer and its location. In most cases, a biopsy is the
only way to definitively diagnose cancer. In the laboratory, cell samples
are examined under the microscope; normal cells look uniform with
similar sizes and orderly organization while cancer cells look less
orderly with varying sizes and without apparent organization. (24)
5) Molecular Diagnosis
Molecular analysis of the oncogenes and tumor suppressor genes
involved in particular types of tumors has the potential of providing
information that is useful in the diagnosis of cancer and in monitoring the
effects of treatment. Molecular diagnostics uncovers the sets of changes
in a normal cell and captures this information as expression patterns. (25)
Microarray analysis is one of the most popular methods initially used to
provide an overview of deregulated genes. (25)
Subsequently, the
expression pattern of potentially interesting genes has to be confirmed by
other methods, such as RT-PCR or northern blot. (26, 27)
Northern blotting, also known as northern hybridization, is a
technique used for detection and quantification of specific RNA levels.
This technique was originally developed by James Alwine and George
Stark, who named it on the basis of its analogy to Southern blotting. (27)
It was the first mRNA detection method used to test gene expression
patterns in human cancer. The identification of altered mRNA levels in
cell lines and tissues leading to overexpression of oncogenes or
downregulation of tumor-suppressor genes reveals one possible initial
event that in addition to mutations sets the cells along the tumorigenic
pathway. (28, 29)
Therefore, the identification of genes differentially
expressed in comparison with normal ‘healthy’ tissue helps to clarify
gene functions.
1- Principle
In northern blot, the sample RNA is separated on the basis of size
with the help of gel electrophoresis. The separated RNA fragments are
transferred to a support membrane and then treated with a labeled DNA
probe. If the sample contains the complementary RNA sequence, the
probe will bind to the membrane and it can be visualized using various
methods (Figure 3). (27)
2- Procedure
The RNA sample is first separated by size using agarose gel
electrophoresis. RNA molecules form streaks rather than bands on
the gel as there are several small fragments of RNA.
The RNA molecules are then transferred to a special blotting paper
usually made of nitrocellulose. Membranes made up of nylon can
Northern Blotting
Technique
also be used. The separation pattern of the RNA molecules in the
blotting paper remains the same as that in the gel.
The blot is then exposed to a labeled, single-stranded DNA probe.
The bases in this probe will pair with their complementary RNA
sequences in the blot producing a double-stranded DNA-RNA
molecule. Although the probe cannot be seen at this stage, since it
is labeled with an enzyme or radioactive tag, it can be seen after
appropriate treatment in the next step.
Next, the probe is exposed to a colorless substrate which gets
converted by the enzyme to a colored product and is visible on an
X-ray film. In case the probe is radioactive, it can directly be seen
on the X-ray film.
Figure 3: Northern Blotting Analysis
3- Applications
Northern blot analysis is widely used in molecular biology as a gold
standard for the direct study of gene expression at the level of mRNA and
to detect transcript sizes. Further applications involve studies of RNA
degradation or RNA splicing as well as RNA half-life. Northern blotting
is also frequently used to check or confirm genetic manipulations in
transgenic or knockout mice. (27)
Advantages and Disadvantages of Northern Blotting
Advantages:
The strength of this method is its simplicity.
Specificity is relatively high.
Sequences with even partial homology can be used as hybridization
probes.
mRNA transcript size can be detected.
RNA splicing is visible because alternatively spliced transcripts
can be detected.
The cost of running many gels is low once the equipment is set up.
Blots can be stored for several years and reprobed if necessary.
Quantity and quality of RNA can be easily verified after
electrophoresis and before further processing is done.
Disadvantages:
Risk of mRNA degradation during electrophoresis: quality and
quantification of expression are negatively affected.
High doses of radioactivity and formaldehyde are a risk for
workers and the environment.
The sensitivity of northern blotting is relatively low in comparison
with that of RT-PCR.
Detection with multiple probes is difficult.
Use of ethidium bromide, DEPC and UV light needs special
training and attention.
VII) Treatment of Cancer
There are many types of cancer treatment. The selection of treatment
and its progress depends on the type of cancer, its locality, and stage of
progression. Surgery, chemotherapy, and radiotherapy are some of the
traditional and most widely used treatment methods. Side effects
associated with traditional methods of cancer treatment highlights the
scope of novel cancer treatment methods. Some of the modern modalities
include hormone-based therapy, anti-angiogenic modalities, stem cell
therapies, immunotherapy and dendritic cell-based immunotherapy.
oncolytic virotherapy, hereditary control of apoptotic and tumor-attacking
pathways, antisense, and RNAi techniques. (30)
1- Surgery
Surgery, resection, or operation is thought as one of the most
promising and conventional treatments of many benign and malignant
tumors as it assures least damage to the surrounding tissues as compared
to chemotherapy and radiotherapy. (31)
2- Radiation Therapy
Radiation therapy is a type of cancer treatment that uses high doses
of radiation to kill cancer cells and shrink tumors. (32)
3- Chemotherapy
Chemotherapy halts tumor progression by killing off their ability to
divide and enforcing apoptosis. Chemotherapy acts here to bring about
changes in the tumor cells so that they stop growing or die; thus, the two
branches of chemotherapeutic drugs are cytostatic (biological drugs) and
cytotoxic, respectively. (33)
4- Hormone Therapy
Advancements in the field of molecular biology in recent years
clarified the role of hormones in cell growth and in the regulation of
malignant cells. Nearly 25% of tumors in men and 40% in women are
known to have hormonal basis. Hormonal treatment is effective to treat
cancer without cytotoxicity which is associated with chemotherapy. (34)
5- Stem Cell Transplant
Stem cell therapeutic strategy is also one of the treatment options for
cancer which are considered to be safe and effective. Application of stem
cell is yet in experimental clinical trial; for example, their use in the
regeneration of damaged tissue like the heart, liver, bones, skin, cornea,
etc. is being explored. Mesenchymal stem cells are currently being used
in trials which are delivered from the bone marrow, fat tissues, and
connective tissues. (35)
6- Anti-angiogenesis therapy
Nutrition to the tumor cells is provided by blood vessels, and the
development of these vessels inside tumor tissues is called angiogenesis.
Some chemical inhibitors known as “angiogenesis inhibitors” can cut off
the blood supply to the tumor cells. These angiogenic inhibitors are
sometimes administered in combination with the chemotherapeutic drugs
in an attempt to increase therapeutic efficiency of both. (36)
7- Autologous dendritic cell vaccines for cancer
immunotherapy
Treatment of chronic infectious diseases or cancer is currently the
main objective of immunotherapy, and it requires better understanding of
the immune systems in terms of its regulatory mechanisms, identification
of appropriate antigen, and optimization of the interaction between
antigen-presenting cells (APC) and T cells. (37)
Dendritic cells are
professional antigen presenting cells (APC). They play a major role in the
initiation and control of immune responses by regulating T and B
lymphocyte activation. These cells are strategically positioned throughout
the body in an immature state, surveying the tissues for invading
pathogens, and are unique in antigen capturing, processing, and
presentation as compared to other antigen-presenting cells. (38)
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