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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