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Laboratory Manual
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Date
The St. Jude Cancer Education Program is an initiative of the Comprehensive Cancer
Center at St. Jude Children’s Research Hospital. Materials may be downloaded free of
charge and reproduced for educational purposes from www.Cure4Kids.org/Kids
© 2010 St. Jude Children’s Research Hospital
St. Jude Cancer Education Program
Damaged Chromosomes and Cancer: the “Philadelphia Chromosome” story
Some Background
In 1914, German scientist Theodor Boveri hypothesized that tumors could be caused by an abnormal number of
chromosomes but he did not have the scientific techniques to prove his theory. Not until the 1950’s were
scientists even able to determine absolutely that the normal number of chromosomes for humans is 46 and to
start defining what is “normal” genetically and what is not. Since then, an even newer field of genetic study has
come into being, called Cytogenetics. Cytogenetics is the study of cell structure and function, especially
chromosomes, and through it scientists have discovered a genetic basis for many diseases, syndromes, and even
some cancers.
Pass It On
When cells replicate as part of mitosis, genetic material is usually passed to the new cells exactly as it existed in
the old cells. In meiosis, half of the material is copied exactly from each parent, and later, halves from two
individuals match up to create a new person. In either case, genes in the DNA can sometimes be altered or
changed, creating a genetic mutation. Some small mutations are harmless and some are actually beneficial,
accounting for how species evolve and adapt. Large-scale mutations can occur where entire chromosomes or
parts of chromosomes can be duplicated incorrectly, be deleted accidentally, or break off and reattach to
another chromosome. This means that there can be extra or missing genes, or new gene combinations, which
can severely affect an individual’s health and development. Genetic mutations can affect a few, many, or all the
cells in an organism, depending on when in life they occur.
Chromosomes and Cancer
In the mid-1950’s, cytogeneticists started studying the chromosomes of patients with cancer and other diseases.
Two young researchers in Philadelphia, Pennsylvania discovered that while many cancer patients with a form of
blood cancer called Chronic Myeloid Leukemia (CML) had the correct total number of chromosomes (46), one
chromosome was abnormally short. Peter Nowell and David Hungeford named the short chromosome the
Philadelphia (Ph) chromosome. It was the first consistent chromosomal abnormality identified in cancer.
Further research now shows that changes in total chromosomal number are not usually associated with most
cancers.
Figure 1. Karyotype of a patient with
CML. This is a photo of the chromosomal
abnormality seen by Nowell and
Hungerford in many patients with CML.
The abnormality is now known as the
Philadelphia (Ph) chromosome.
High School Lab Exercise, Module 1
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Improved cytogenetic techniques in the 1970s showed that the Ph chromosome is the result of a translocation
between chromosomes 9 and 22. A translocation is when a piece of one chromosome breaks off and attaches to
another chromosome. Scientists don’t fully understand what causes the translocation to occur. In theory, they
can occur during mitosis or meiosis in any cell at any stage of development. The cell type, stage of growth and
the genes involved in a translocation all influence how slight or severe the change may be.
In the late 1970s and early 1980s, scientists began using new technologies to look for what specific genes were
altered in human cancers. In the case of CML, it was discovered that the critical genes involved in this
translocation were “ABL” on chromosome 9 and “BCR” on chromosome 22.
Figure 2. Translocation results in a new BCR-ABL gene.
The Ph chromosome is the result of the translocation of
the ABL gene on chromosome 9 onto the BCR gene on
chromosome 22, leading to the formation of the new
BCR-ABL gene.
Translocation
Normal
Abnormal
Later, it was found that the new BCR-ABL gene leads to an abnormal cellular signal that causes cells to grow and
divide continuously. This signal cannot be turned off the way it can be in normal cells. The BCR-ABL gene is part
of a class of genes called oncogenes. Oncogenes result from one or more mutations in a gene that normally
promotes normal cell growth and division. It is the uncontrolled cell division caused by the BCR-ABL oncogene
that produces CML.
Figure 3. The Ph chromosome and leukemia.
Expression of the BCR-ABL gene from the Ph
chromosome in the hematopoietic stem cells is the
cytogenetic hallmark of CML, which results in the
accumulation of granulocytes in the blood.
The Ph chromosome translocation is present in more than 90% of patients with CML. The disease is usually
symptom-free or very mild in its first phase. However, CML rapidly progresses to an accelerated phase. In the
past, once the accelerated phase was reached, patients generally died within 6-12 months despite stem cell
transplants and aggressive chemotherapy regimens.
High School Lab Exercise, Module 1
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In the 1990s, a major discovery was made in terms of chemotherapy for CML. A newly developed therapeutic
agent called imatinib mesylate was found to stop the cellular growth signal coming from the BCR-ABL gene. By
blocking the signal, the drug reduces the abnormal effects of the Ph chromosome. In addition, the drug can also
cause direct death of the abnormal cells that express the BCR-ABL gene.
A.
Uncontrolled
Cell Growth
BCR-ABL
Protein
B.
Imatinib
Mesylate
BCR-ABL
Protein
Blocks Abnormal
Cell Growth
Figure 4. Mode of action of
imatinib.
A. In patients with CML, the
BCR-ABL protein is part of a
signal pathway that causes
uncontrolled cell growth.
B. When the drug imatinib is
introduced, the cellular growth
signal pathway is interrupted.
Abnormal cells no longer grow
uncontrolled.
Patients can take imatinib for as long as the disease continues to respond and as long as they are able to
tolerate any side effects, which are generally mild. This is good news for the many patients who will now live
longer thanks to the scientists and doctors who worked hard to find this treatment.
Doctors and scientists at St. Jude Children’s Research Hospital are studying genetic abnormalities related to
cancer every day in the pursuit of better treatments and cures for childhood cancers. A St. Jude scientist helped
discover the specifics of the BCR-ABL gene and how it relates to CML. The scientist continues to work with the
Ph Chromosome and other chromosomal problems that play a role in childhood cancer. Other groups of St. Jude
doctors and scientists are trying to determine which specific genetic mutations occur in different cancers and
how specific genetic mutations influence the effectiveness of certain treatments. The discoveries made at St.
Jude lead to new treatments and cures and give hope to the families and patients affected by childhood cancer.
Review Questions
1. How many (total) chromosomes are in a normal human cell?
4
2. What type of mutation causes the abnormal Philadelphia chromosome to appear in a human?
Translocation
3. What happens in abnormal cells that have the BCR-ABL gene?
Too much cell division
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
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Laboratory Investigation: Chromosomes and Leukemia
Introduction to Chromosomal Banding
Did you know that the hereditary nature of every living organism is defined by its genome? The genome consists
of long sequences of DNA that provide the information needed to construct an organism. If you were to line up
the DNA from just one of your cells end-to-end, it would be over 7 feet long. That’s about 80 billion miles of DNA
from all the cells in an average adult human!
A human genome can be divided into chromosomes. There are 23 pairs of chromosomes in every human cell
(remember, we acquire one set of 23 chromosome from each parent through meiosis). Chromosomes can be
divided into genes. A gene is a sequence of DNA that contains information to perform a cellular function. There
are over 30,000 genes in the human genome, and a copy of the entire genome is present in the nucleus of every
cell of the body (with a few exceptions—such as red blood cells and platelets).
Chromosomes can only be seen during cell division because they condense. Seeing chromosomes requires a
special stain and a microscope. One technique that cytogeneticists use to study chromosomes is called
chromosomal banding, or G-banding. G-banding is the conventional means of staining chromosomes to reveal
their characteristics. Once stained, a standard light microscope is used to view the chromosomes. This technique
is called G-banding because the stain, or dye, used is called Giemsa. When stained, the chromosomes look like
strings with light and dark bands. The stain binds with gene-poor areas of the chromosome, so the dark bands
represent the parts of the chromosome that do not have very many genes. This technique allows scientists to
identify the different chromosomes and detect the presence of any obviously abnormal chromosomes.
Each chromosome has two “arms”, separated by a pinched area in the center called the centromere. The short
top arm is called the p (petit) arm, and the long bottom arm is called the q (next letter in the alphabet) arm. The
first 22 pairs of chromosomes are numbered from longest to shortest, while the last pair, called the sex
chromosomes, are labeled X or Y. Females have two X chromosomes (XX), and males have one X and one Y
chromosome (XY). Cytogeneticists can create a karyotype of all the chromosomes from one cell and analyze an
enlarged photo of it to identify any chromosomal abnormalities. A karyotype is a common way to look at
chromosomes for analysis. In a karyotype, chromosomes are lined up numerically in pairs from longest to
shortest based on banding pattern. Chromosomes of the white blood cells are used to create karyotypes since
they can be easily isolated from a vial of blood.
Figure 5. Making a Karyotype.
Cytogeneticists use a small amount of
a patient’s blood cells for the
karyotype. Cells grow in a Petri dish
and go through mitosis. Mitosis is
stopped in metaphase with
chemicals. Chromosomes are viewed
and photographed under the
microscope. Chromosomes are cut
out from the photo and arranged into
a karyotype.
High School Lab Exercise, Module 1
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Chromosome length,
NOT necessarily shape
Centromere location
Band pattern
Figure 6. Normal Male Karyotype. Cytogeneticists use
the length of the chromosome, the band pattern and the
location of the centromere to match and make pairs. A
female would have two Xs instead of one X and one Y. In
a karyotype the chromosomes can appear bent or
twisted. This is normal and simply reflects how they are
sitting on the slide.
Using Chromosomal Banding to Diagnose CML—Can you do it?
Scenario:
A man and a woman came to a hospital on the same day for some blood tests. A different nurse saw
each patient. The man and woman have the same last name. Both nurses labeled the sample with the
patients’ last name, Smith, and now they don’t know which sample is the man’s and which is the
woman’s.
The hospital needs your help figuring out which results are which. You will receive two sets of
chromosomes—one from each patient. Please identify which set, A or B, goes with the female
patient and which goes with the male patient. Please also note if you see any chromosomal
abnormalities in either patient’s DNA. Give your recommendations for a diagnosis if you can.
Results:
Patient A: Smith, M
Male
Chromosomal abnormalities: Yes
Comments: Students should note that the homologous pairs and chromosomes 9 and 22 do not match
one another. They should also notice that the patient lacks a second X chromosome.
Patient B: Smith, M
Female
Chromosomal abnormalities: No
Comments: Students should note that all homologous pairs have matching banding patterns.
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
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Glossary
Centromere—an extra-condensed region of the
chromosome where sister chromatids connect
Chemotherapy – strong chemical drugs used to
treat cancer
Chromosomal banding—common technique used
to stain and study chromosomes under a light
microscope
Chromosomes—string-like strands of DNA found in
a cell’s nucleus that contain genes
Chronic myeloid leukemia (CML) – a specific type of
cancer in which too many mature white blood cells
are produced and released in the blood. CML is
more common in adults than in children. About 1-2
in 100,000 people over the age of 45 develop CML
Cytogenetics – the study cell structure and function,
especially of chromosomes
Gene—a sequence of DNA that contains
information to perform a cellular function
Karyotype – a common way to visualize
chromosomes for analysis; chromosomes are lined
up numerically in pairs
Leukemia – a class of cancer of the blood cells
Meiosis—a two-part cell division that occurs in
organisms that reproduce sexually; gametes are
produced and contain half of the organism’s genetic
material
Metaphase—the second stage of cell division
Mitosis—cell division where one cell becomes two;
the two resulting daughter cells are identical to the
parent cell
Mutation—a permanent change in the structure of
an organism’s DNA
Oncogene—results from one or more mutations in
a gene that normally promotes normal cell growth
and division
Genome—consists of long sequences of DNA that
provide the information needed to construct an
organism
Philadelphia (Ph) chromosome – a small
chromosome resulting from a translocation
between human chromosomes 9 and 22; present in
more than 90% of patients with CML
Giemsa – a chemical dye used commonly in the
staining of blood and chromosomes
Proliferation – cell growth and reproduction;
increase in cell number
Granulocyte – a class of white blood cells; includes
neutrophils, basophils, etc.
Translocation – when a piece of one chromosome
breaks off and attaches to another chromosome
Hematopoietic stem cell – a cell that can develop
into any specialized type of blood cell
White blood cells – a class of cells responsible for
fighting infections; includes neutrophils,
lymphocytes, macrophages, etc.
Imatinib mesylate– a chemotherapy drug used to
treat CML
A special thanks to Dr. Charles Mullighan and Dr. Racquel Collins-Underwood in the Pathology Department at St. Jude Children’s
Research Hospital for their help in producing this lab manual.
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
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Patient A Chromosomes
Below is a set of 23 chromosomes. Cut out the chromosomes and paste them next to their matching
homologous pair to complete the karyotype.
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
www.Cure4Kids.org/Kids
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Patient A Karyotype
High School Lab Exercise, Module 1
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Patient B Chromosomes
Below is a set of 23 chromosomes. Cut out the chromosomes and paste them next to their matching
homologous pair to complete the karyotype.
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
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Patient B Karyotype
High School Lab Exercise, Module 1
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Patient A: Answer Key
Once students have completed their karyotypes for both patients, pass around the answer key and
have them check their work by noting the number they got correct and the number they missed.
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
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Patient B: Answer Key
Once students have completed their karyotypes for both patients, pass around the answer key and
have them check their work by noting the number they got correct and the number they missed.
High School Lab Exercise, Module 1
© 2010 St. Jude Children’s Research Hospital
www.Cure4Kids.org/Kids
Page 13 of 13