Molecular Cytogenetics

Methods

Flowcytometric and FISH Markers in

Childhood Acute Lymphoblastic Leukemia
Dr Gihan EL Hussieni Gawish, MSc, PhD
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Acknowledgements
First of all, I cannot give a word to fulfill my deeps love and thanks
to (Allah) for lighting me the way not only throughout this piece of work
but also throughout my whole life.

This work is dedicated to:

My husband; Dr Hussein Al Omer and my Family

I am indebted to King Saud University for support and encouragement to

finish this work.
Also, I wish to express my deep thanks to:
 Prof. Dr./ Abdelfattah M. Attalah, Professor of Genetics &
Immunology, George Washington University, USA (Former).
Director of Biotechnology Research Center, New Damietta, Egypt,
for continuous advice
 Prof. Dr./ Ahmed Abd Al Salam Settin, Professor of Pediatrics &
Genetics, Faculty of Medicine, Mansoura University for his
continuous help

Finally, I am indebted to all the team of the honorable Genetics

Unit, Mansoura University Children Hospital, for their continuous
support and encouragement

Gihan El Hussieny Gawish

January 2009
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Contents
Title Page
Introduction 1
Review of Literature 4
I- Acute lymphoblastic leukemia 4
1-Definition 4
2-Incidence of Leukemia 5
3-Types of leukemia 6
4-Biological Classification of ALL 6
5-Causing of Leukemia 7
6-The Signs of Leukemia 9
7-Stages of Childhood ALL 11
8-Treatment of Childhood ALL 11
9-Four Phases of Treatment 14
II- Cell cycle and apoptosis 15
1-Cell cycle 15
1-1-Cell cycle and cancer 19
2-Apoptosis and its markers 20
2-1-The mechanism of apoptosis 21
2-2-Apoptosis-targeted therapies for hematologica malignancies 25
2-3-The apoptosis promoter (p53) 30
2-4-The inhibitor of apoptosis 35
2-4-1-Bcl2 proteins 35
2-4-2-C-myc oncogene 38
III-Flow cytometry 42
1-Introduction 42
2-Principles of flow cytometric instrumentation 44
2-1-Fluidic system 46
2-2-Illumination system 49
2-3-Optical and electronics system 53
2-4-Data storage and computer control system 54
3-Data analysis 59
63
IV- Applications of flow cytometry
1-Cell cycle analysis 65
1-1-Staining procedure 66
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Page
1-2-Evaluation of DNA histogram 67
2-Immunophenotyping Applications 71
2-1-Erythrocyte analysis 72
2-2-HIV monitoring 73
2-3-Immunophenotyping of leukemias 73
2-4Quantification of stem cells 75
2-5-Platelet analysis 75
2-6-Testing for HLA-B27 76
3-Major applications of apoptosis analysis 76
3-1- Apoptosis light scatter 77
3-2-Apoptosis DNA analysis 78
3-3-Apoptosis cell membrane analysis 80
3-4-Apoptosis enzyme analysis 82
3-5-Apoptosis organelle analysis 83
4- Detection of apoptotic markers 83
86
V- Flourescence in situ hybridization
1-Introduction 86
2-Three different types of FISH probes 91
2-1-Locus specific probes 91
2-2-Alphoid or centromeric repeat probes 91
2-3-Whole chromosome probes 92
3-Applications of FISH 92
3-1-ALL investigation by FISH 96
3-1-1-Philadelphia 97
VI- References 106
VII- Life Flowcytometric Figures 144
VIII-Life FISH Pictures 147
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List of Figure
Review of Literature Page
Figure (2-1) A schematic representation of the mammalian cell cycle 18
Figure (2-2) The intrinsic or mitochondrial pathway 22
Figure (2-3) The mechanism of apoptosis (Apoptosis triggered by 24
external signals: the extrinsic or death receptor path way)
Figure (2-4) Diagram of the mitochondrial and death receptor pathways 26
of cell death
Figure (3-1) Facscaliblur flow cytometry instrument 47
Figure (3-2) Flow cytometer system (Facscalibur) 48
Figure (3-3) Flow cytometers use the principle of hydrodynamics 50
focusing for presenting cells to a laser

Figure (3-4) A simplified illustration of Flow Cytometry 52

Figure (3-5) Two parameter histogram and dot plot displaying FL1-FITC 57
on the x axis and FL2-PE on the y axis
Figure (3-6) FlowJo program 58
Figure (3-7) Analysis pulse width versus pulse height or area we can 60
eliminate the majority of G0 doublets that appear as G2
Figure (3-8) DNA histogram 61
Figure (3-9) DNA histogram (aneuphliod population) 62
Figure (4-1) Coeffecient of Variation (C.V.) 69
Figure (4-2) Propidium iodide and TO-PRO-3 79
Figure (4-3) Sub G1 peak by propidium iodide staining 81
Figure (5-1) Fluoresence in situ hypridization 88
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List of Abbreviations

AL Acute leukemia
ALL Acut lymphoblastic leukemia
ALT Alanine amino transaminase
AO Acridine orange
AST Aspartate amino transaminase
BM Bone marrow
CBC Complete blood picture
CD Cluster of differentiation
CML Chronic myeloid leukemia
CV Coefficient of variation
DAPI 4-6 diamino -2-2phenylindole
DI DNA index
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
EB Ethedium bromide
EDTA Ethyline diamine tetraacetic acid
FAB French American British
FACS Flow activated cell sorter
FISH Fluorescence in situ hybridization
FITC Flourecien isothiocyanate
G0/G1 Phase represents the gap in of DNA replication time
between mitosis and the start
G2/M Phase represents the gap between the end of DNA
replication onest of mitosis
HIV Human immunodeficiency virus
HLA Human leukocyte antigen
HPV Human papilloma virus
LC Liver cirrhosis
MMC Mithramycin
MRD Minimal residual disease
PBS Phosphate buffer saline
PI Propidium iodide
PS Phosphatidylserine
S phase DNA syntheis
WBC White blood cells
 7

Introduction

Childhood acute lymphoblastic leukemia (ALL) is a disease in

which too many underdeveloped lymphocytes are found in a child's blood
and bone marrow. Lymphocytes are infection-fighting white blood cells.
ALL is the most common form of leukemia in children, and the most
common kind of childhood cancer (Moorman et al., 2006).

Acute lymphoblastic leukemia (ALL) represents nearly one third of

all pediatric cancers. Annual incidence of ALL is about 30 cases per million
populations, with a peak incidence in patients aged 2-5 years. Although a
small percentage of cases are associated with inherited genetic syndromes,
the cause of ALL remains largely unknown (Jeffrey, 2005).

Flow cytometry can be applied in basic research and in the clinic to

identify and measure apoptotic cells. The choice of a particular flow method
depends on several variables (cell system, type of flow cytometer, type of
apoptosis inducer, type of information required) (Bogh and Duling, 2005).

The cell cycle was subdivided into four consecutive phases; G1 or

pre-synthetic phase, S, G2 or post-synthetic phase, and M phase during which
mitotic division into two daughter cells takes place. The G2 phase represents
the gap in time between the end of DNA replication and onset of mitosis. It is
possible to discrimination between G1 vs, S vs, G2 or M cells because of the
difference in their DNA content (Rabinovitch, 1993).

The DNA content of the cell can provide a great deal of information
about the cell cycle. The measurement of the DNA content of cells was one
of the first major applications of flow cytometry (Albro et al., 1993).
 8

Apoptosis (programmed cell death) is a physiologic phenomenon

where in the dying cell plays an active part in its own destruction (Schuler
et al., 1994). Apoptosis plays a role in many diseases. There is a great
potential for treatment of these diseases in developing agents that can alter
the apoptotic process and change the natural disease progression. Molecules
whose roles in apoptosis have been investigated include Bcl-2 and c-myc
proteins, the p53 tumor suppressor gene and various tumor suppressor gene
products (Menendez et al., 2004).

P53 is a pro-apoptotic genes present in all cells, but has special

significance to cancer cells. It is a tumor repressor gene, meaning that its
presence reduces the occurrence of cancer tumors by promoting apoptosis in
cancer cells (Polyak et al., 1997). BCL2 is an important regulator of
apoptosis, The oncogenic activity of the Bcl2 gene is carried out via
suppression of lymphocytic apoptosis or programmed cell death (Cory &
Adams, 2002 and Roumier et al., 2002). C-Myc is widely known as a crucial
regulator of cell proliferation in normal and neoplastic cells (Wechsler et al.,
1997& Facchini and Penn, 1998).

The technology of flow cytometry and the discovery of a method to

produce monoclonal antibodies have made possible the clinical use of flow
cytometry for the identification of cell populations. Monoclonal antibodies
(tagged) with the fluorescent dye are commonly used for the identification of
cell surface antigens and fluorescent dyes that directly and specifically bind
to certain components of the cell (i.e. DNA) are used for cell cycle analysis
(Zhang et al., 2005).

Fluorescence in situ hybridization (FISH) allows identification of

specific sequences in a structurally preserved cell, in metaphase or interphase
 9

(Chatzimeletiou et al., 2005). FISH is increasingly used for the identification

of ALL. FISH plays an important role in detecting chromosome changes
(Primo et al., 2003).

Almost all the chromosome abnormalities in ALL are translocations.

The most common one is Philadelphia chromosome. It is the main product of
the t(9;22) translocation. This translocation causes a rearrangement between
the proto-oncogene c-ABL and a gene called the breakpoint cluster region
(BCR). The BCR/ABL fusion gene resulting from t(9;22) translocation. FISH
is increasingly used for the identification of BCR/ABL gene rearrangements
(Rudolph et al., 2005).

І- Acute Lymphoblastic Leukemia

 10

Acute lymphoblastic leukemia (ALL) is the most common form of

childhood cancer. It is a type of cancer that starts from white blood cells in
the bone marrow called lymphocytes. In most cases it quickly moves into the
blood. It can then spread to other parts of the body including the lymph
nodes, liver, spleen and central nervous system (Moorman et al., 2006).

Leukemia is a cancer of the blood cells. There are several types of

leukemia and these are classified by how quickly they progress and what cell
they affect. Acute leukemia is fast-growing and can overrun the body within
a few weeks or months. By contrast, chronic leukemia is slow-growing and
progressively worsens over years (Carolyn et al., 2002).

Normal blood cells contain white blood cells, red blood cells,
platelets and fluid called plasma. All of these products are formed in the bone
marrow, a spongy area located in the center of bones. It contains a small
percentage of cells that are in development and are not yet mature. These
cells are called blasts. Once the cell has matured, it moves out of the bone
marrow and into the circulating blood. The body has mechanisms to know
when more cells are needed and has the ability to produce them in an orderly
fashion (Carroll et al., 2003).
 11

1-Incidence of Leukemia:

Acute lymphoblastic leukemia is the most common form of

childhood leukemia where it accounts for about 75% of childhood leukemia
and 25% of all pediatric cancer (Lanzkowsky, 2000). National Cancer
Institue, Cairo University, ALL represents 23.3% of all pediatric
malignancies and 75% of all pediatric leukemias. In a more recent research in
the Pediatric Heamatology/Oncology Unit, Ain Shams University Hospital,
ALL constitutes 82% of all leukemic cases (Khalifa et al., 1999).

The global incidence of leukemias is about 8 to 9 per 100,000 people

each year. Approximately 250,000 new cases occur annually worldwide.
Leukemia accounts for 2.5% of overall cancer incidence. However, its
incidence among children demonstrates its significance. Although childhood
cases (through 14 years of age) account for about 12% of all leukemias,
childhood cancer is the second biggest killer of children (after accidents) and
leukemia is the most common form of childhood cancer. The incidence of
childhood ALL in the United States has increased approximately 20% over
the past two decades, mostly in the 0- to 4-year-old age group. Over the
course of this century, leukemia rates have also generally increased (Sandler
and Ross, 1997).

Acute lymphoblastic leukemia affects slightly more boys than girls.

It occurs predominantly in children, peaking at four years of age. It is seen
more frequently in industrialized nations, and it is slightly more common
among white children and boys. Studies have suggested that patients who are
younger than thirty five years of age far better than older patients (Jeffrey,
2005).
 12

2-Types of leukemia:
By considering whether leukemias are acute or chronic, and whether
they are myelogenous or lymphocytic, they can be divided into four main
types. The first one is an acute myeloid leukemia which occurs in both
children and adults. The second one is an acute lymphocytic leukemia which
is the most common type seen in children, but also seen in adult's over65.The
third one is a chronic myelogenous leukemia which occurs mostly in adults.
Chronic lymphocytic is the fourth type which is the most often seen in people
over age55, can affect younger adults, but almost never seen in children (Pui,
1995).

In acute leukemia, the bone marrow cells are unable to properly

mature. Immature leukemia cells, which are often called blasts, continue to
reproduce and accumulate. In chronic leukemia, the cells can mature but not
completely. They are not really normal. They generally do not fight infection
as well as do normal white blood cells. Of course, they live longer, build up,
and crowd out normal cells. The types of leukemia are also grouped by the
type of white blood cell that is affected, leukemia that affects lymphoid cells
is called lymphocytic leukemia, and leukemia that affects myeloid cells is
called myeloid leukemia or myelogenous leukemia (Lichtman et al., 1995).

:Biological Classification of ALL-3

Acute lymphoblastic leukemia blasts are derived from either B-cell
or T-cell lineages, as determined by cell surface and other markers. A small
percentage of the cells are either so primitive that they do not express enough
markers to identify (Ross et al., 2003 and Pullen et al., 1999).
 13

Acute lymphoblastic leukemia is categorized according to a system

know as the French-American-British (FAB) Morphological Classification
Scheme for ALL. ALL1 is mature-appearing lymphoblasts (T-cells or pre-B-
cells); these cells are small with uniform genetic material, regular nuclear
shape, nonvisible nucleoli, and little cytoplasm. ALL2 is immature and
pleomorphic lymphoblasts (T-cells or pre-B-cells), these cells are large,
variable in size, varaiable genetic material, irregular nuclear shape, one or
more large nucleoli and variable cytoplasm. ALL3 is lymphoblast(B-
cells),these are large, genetic material is finely stripped and uniform, nuclear
shape is regular, there are one or more prominent nucleoli, and cytoplasm is
moderately abundant (Schrappe et al., 2000).

4-Causing of Leukemia:
The causes of the disease are not known, but experts believe that
ALL develops from a combination of genetic and environmental factors. A
number of genetic mutations associated with ALL have been identified.
Missing or defective genes that suppress tumors are responsible for cases of
ALL (Guo et al., 2005).

Several things have been identified as risk factors-that is, exposure

to them puts a person at a higher risk of developing leukemia, but it is not a
certainly that this exposure will lead to leukemia. These factors include
exposure to high-energy radiation, like that released from a nuclear accident
or bomb. Some genetic syndrome put a person at higher risk. People who
work with the chemical benzene over a long period of time also have a
greater chance of getting leukemia. Some scientist feel that exposure to
electromagnetic fields, like those that come from power lines, may put a
person to higher risk, but this has not been proven (Pui et al., 2001).
 14

Heredity, radiation, chemical exposures, and treatment with

chemotherapeutic agents have been implicated in the development of
leukemia. Viral infection by at least one known virus, human T-cell
leukemia/lymphotropic virus type I (HTLV-1), is a well-understood cause of
adult T-cell leukemia (Franchini, 1995 and Greaves, 1997).

Another group of risk factors includes occupational and

environmental exposure to radiation or chemicals. The best established cause
of leukemia among children is in utero exposure to diagnostic X-rays.
Leukemia in adults is strongly associated with occupational exposure to
ionizing radiation. There is little evidence, however, that nonionizing
radiation such as electromagnetic fields (EMF) induces leukemia. Indeed,
two recent studies have shown that EMF exposure is not a major risk factor
for leukemia in children or in adults. Some studies have reported an
association between cancer and high levels of electromagnetic radiation
(EMR). Whether lower levels of radiation (eg, living near power lines, video
screen emissions, small appliances, cell phones) play any major role is
uncertain but probably unlikely (Linet et al., 1997 and Verkasalo, 1996).

Because most people in the general population are not exposed to

chemotherapeutic drugs or occupationally exposed to radiation or chemical
solvents, exposure to these agents cannot explain the causes of the majority
of leukemia cases diagnosed each year. We conservatively estimate that the
causes of at least 20,000 (approximately 70%) of the 28,000 new leukemia
cases that develop annually in the United States are unexplained. Thus, the
causes of leukemia remain largely unknown. Although some success has
been achieved in treating leukemias, especially in children, mortality rates
have remained relatively high (approximately 75% in the United States)
(Kazak et al., 1997).
 15

Genetic predisposition may play a major role in both adult and

childhood leukemia. Although the Leukemia Society of America emphasizes
the fact that anyone may develop the disease, an increased risk exists among
Eastern European Jews, and a decreased risk exists among Asians
(differences in diet and lifestyle may play a role, however). Individuals with a
family history of leukemia or lymphoma have a 5.6-fold increased risk for
AML. Parents of children with Down syndrome also have an increased risk
of leukemia (Greaves, 1997 and Shannon et al., 1992).

Up to 65% of leukemias contain genetic rearrangements, called

translocations, in which some of the genetic material (genes) on a
chromosome may be altered, or shuffled, between a pair of chromosomes.
For example the most common genetic injury in ALL is t(12;21), which
means a translocation with a genetic shift between chromosome 12 and 21. It
is also referred to as TEL-AML1 fusion and occurs in approximately 20% of
ALL patients. Researchers believe that this translocation may occur during
fetal development in some patients. About 20% of adults and about 5% of
children with ALL have a genetic abnormality called the Philadelphia (Ph)
chromosome t(9;22). Another important chromosome translocation is t(4;11)
involving the MLL gene on chromosome II. Often occurring in children
under one year old (Khandakar et al., 2005).

:The Signs and diagnosis of Leukemia-5

The blast cells are unable to perform their normal function of
fighting infection, so patients may develop fevers or infections that won't go
away. As the number of immature cells (blasts) increases, the normal cells
are crowded out. This leads to low red blood cell counts and platelets (Smith
et al., 1996).
 16

Acute lymphoblastic leukemia tends to cause symptoms more rapidly

than chronic leukemia. Some common symptoms include fever, chills,
weakness and fatigue, swollen or tender lymph nodes, liver or spleen, easy
bleeding or bruising, swollen or bleeding gums, night sweats, and bone pain.
The abnormal cells can accumulate in the brain or spinal cord, causing
headaches, vomiting, confusion, or seizures (Adachi et al., 2005).

In acute lymphoblastic leukemia, the doctor asks about medical

history and conducts a physical exam. During the exam, abnormalities such
as enlarged spleen, liver or lymph nodes may be detected, prompting further
investigation. Complete blood count would find blast cells present in the
blood, thus suggestion a diagnosis of leukemia. This test can reveal that the
patient has leukemia. A sample of bone marrow is determined the type of
leukemia (Champlin et al. 1989 and Burger et al., 2003).

A complete blood cell count is the first step in diagnosing ALL. This
test will often show various findings, including the following: The presence
of circulatory leukemic blast cells, the presence and severity of anemia and
the count of a variety of blood cell types. (A high white blood cell count
indicates a more severe disease.) These tests will not always show the
presence of leukemic cells. Blood tests do not always detect leukemia, and
about 10% of patients with ALL have a normal blood cell count (Adachi et
al., 2005).

If the results of the blood tests are abnormal or the physician

suspects leukemia despite normal cell counts, a bone marrow aspiration and
biopsy are the next steps (Rezaei et al., 2003).
 17

If bone marrow examination confirms ALL, a spinal tap may be

performed, which uses a needle inserted into the spinal canal. A sample of
cerebrospinal fluid with leukemia cells is a sign that the disease has spread to
the central nervous system. In most cases of childhood ALL, leukemic cells
are not found in the cerebrospinal fluid (Vieira et al., 2005).

:Treatment of Childhood ALL-6

The treatment depends on age, the results of laboratory tests, and
whether or not the child has been previously treated for leukemia. Untreated
ALL means that no treatment has been given except to reduce symptoms.
There are too many white blood cells in the blood and bone marrow, and
there may be other signs and symptoms of leukemia. Remission means that
treatment has been given and the number of white blood cells and other blood
cells in the blood and bone marrow is normal that there no signs or symptoms
of leukemia. Recurrent disease means that the leukemia has come back after
going into remission. Refractory disease means that the leukemia failed to go
into remission following treatment (Bassan et al., 1997).

There are treatments for all patients with childhood acute

lymphoblastic leukemia. The primary treatment for ALL is chemotherapy.
Radiaion therapy may be used in certain cases. Bone marrow transplantation
is being studied in clinical trials (Uckun et al., 1997).

Acute lymphoblastic leukemia patients should receive chemotherapy

drugs as soon as possible after diagnosis. Chemotherapy uses strong drugs to
kill leukemia cells. The goal of chemotherapy is to achieve remission (no
symptoms of ALL) and to restore normal blood cell production. Common
chemotherapy drugs include doxorubicin, fludarabine and cyclophosphamide.
 18

The drugs used depend on factors such as the patient's age and the number
and type of leukemia cells in the blood. Unfortunately, chemotherapy also
kills normal cells, so ALL patients receiving chemotherapy may have side
effects, including nausea, tiredness and a higher risk of infections (Balduzzi
et al., 2005).

For most patients, chemotherapy restores normal blood cell

production within a few weeks, and microscopic examinations of their blood
and marrow samples will show no signs of leukemia cells. When this
happens, the disease is in remission. Although chemotherapy often brings
long-lasting remissions in children, in adults, ALL frequently returns. If the
ALL returns, patients and their doctors can consider more chemotherapy or a
marrow or blood cell transplant. Chemotherapeutic agents kill cancer cells by
activating apoptosis, or programmed cell death. Major apoptotic pathways
and the specific role of key proteins in this response is described. The
expression level of some of these proteins, such as Bcl2, BAX, and caspase 3,
has been shown to be predictive of ultimate outcome in hematopoietic
tumors. New therapeutic approaches that modulate the apoptotic pathway are
now available and may be applicable to the treatment of childhood ALL
(Donadieu & Hill, 2001 and Nakase et al., 2005).

Radiation therapy uses X-rays or other high-energy rays to kill

cancer cells and shrink tumors. Radiation for ALL usually comes from a
machine outside the body (external beam radiation therapy) (Durrant et al.,
1997).

Bone marrow transplantation is a newer type of treatment. First, high

doses of chemotherapy with or without radiation therapy are given to destroy
all of the bone marrow in the body. A bone marrow transplant using marrow
 19

from a relative or person not related to the patient is called an allogeneic bone
marrow transplant (Ulrich et al., 2001).

An even newer type of bone marrow transplant, called autologous bone

marrow transplant, is being studied in clinical trials. During this procedure,
bone marrow is taken from the patient and may be treated with drugs to kill
any cancer cells. The marrow is frozen to save it. The patient is then given
high-dose chemotherapy with or without radiation therapy to destroy all of
the remaining marrow. The frozen marrow that was saved is thawed and
given through a needle in a vein to replace the marrow that was destroyed
(Sebban et el., 1994).

Treatment outcome is dependent not only on the therapy applied, but

importantly, also on the underlying biology of the tumor and the host. Each of
these variables must be factored into initial treatment decisions, as well as
later refinements based on initial response, and several biological features. It
is recognized that with improvements in therapy, certain variables might lose
their prognostic value; therefore, risk assignment plans should be routinely
reassessed. Finally an optimal system should allow for comparison of the
outcomes of similar or identical patients, treated on different protocols (Choi
et al., 2005).

There are generally four phases of treatment for ALL. The first
phase, remission induction therapy, uses chemotherapy to kill as many of the
leukemia cells as possible to cause the cancer to go into remission. The
second phase, called central nervous system (CNS) prophylaxis, is preventive
therapy, it involves using intrathecal and/or high-dose systemic
chemotherapy to the CNS to kill any leukemia cells present there. It is also
 20

used to prevent the spread of cancer cells to the brain and spinal cord even if
no cancer has been detected there. Radiation therapy to the brain may also be
given, in addition to chemotherapy, for this purpose. CNS prophylaxis is
often given in conjunction with consolidation therapy. Once a child goes into
remission and there no signs of leukemia, a third phase of treatment called
consolidation or intensification therapy, is given. Consolidation therapy uses
high-dose chemotherapy to attempt to kill any remaining leukemia cells. The
fourth phase of treatment, called maintenance therapy, uses chemotherapy for
several years to maintain the remission (Attal et al., 1995).
 21

II- Cell cycle and Apoptosis

1-Cell cycle:
The concept of the cycle in its current form is introduced by
Howard and Plec, (1953). They observed that DNA synthesis (S- phase) in
individual cells was discontinuous and occupied a discrete portion of the cell
life and was constant in duration. Mitotic division (M-phase) was seen to
occur after certain period of time following DNA replication. A distinct
phase between DNA replication and mitosis was also apparent (Look et al.,
1996).

Cell cycle phase of G1 was historically considered to be a time cells

had little observable activity. Since this time precedes DNA synthesis, the
term Gap 1 (G1) was coined. They have diploid chromosome (2C=46
chromosome). At a certain point in the cell's life, the DNA synthetic
machinery turns on. This phase of the cell's life is labeled "S" for synthesis.
As the cell proceeds through this phase, its DNA content increases from 2C
to 4C. At the end of S, the cell has duplicated its genome and it is in the
tetraploid state. After the S phase, the cell again enters a phase that was
historically thought to be quiescent. Since this phase is the second gap region,
it is referred to as G2. In the G2 phase, the cell is producing the necessary
proteins that will play a major role in cytokinase. After a highly variable
amount of time, the cell enters mitosis (M). DNA content remains constant at
4C until the cell actually divides at the end telophase (Liblit, 1993).

The process of replicating DNA and dividing a cell can be described

as a series of coordinated events that compose a "cell division cycle,"
 22

illustrated for mammalian cells in Fig (2-1). In each cell division cycle,
chromosomes are replicated once (DNA synthesis or S-phase) and segregated
to create two genetically identical daughter cells (mitosis or M-phase). These
events are spaced by intervals of growth and reorganization (gap phases G1
and G2). Cells can stop cycling after division, entering a state of quiescence
(G0). Commitment to traverse an entire cycle is made in late G1. At least two
types of cell cycle control mechanisms are recognized: a cascade of protein
phosphorylations that relay a cell from one stage to the next and a set of
checkpoints that monitor completion of critical events and delay progression
to the next stage if necessary (Nasmyth, 1996).

The first type of control involves a highly regulated kinase family.

Kinase activation generally requires association with a second subunit that is
transiently expressed at the appropriate period of the cell cycle; the periodic
"cyclin" subunit associates with its partner "cyclin-dependent kinase" (CDK)
to create an active complex with unique substrate specificity. Regulatory
phosphorylation and dephosphorylation fine-tune the activity of CDK-cyclin
complexes, ensuring well-delineated transitions between cell cycle stages
(Elledge, 1996).

A second type of cell cycle regulation, checkpoint control, is more

supervisory. It is not an essential part of the cycle progression machinery.
Cell cycle checkpoints sense flaws in critical events such as DNA replication
and chromosome segregation. When checkpoints are activated, for example
by underreplicated or damaged DNA, signals are relayed to the cell cycle-
progression machinery. These signals cause a delay in cycle progression, until
the danger of mutation has been averted. Because checkpoint function is not
required in every cell cycle, the extent of checkpoint function is not as
 23

obvious as that of components integral to the process, such as CDKs (Sherr,

1996).

Figure (2-1): A schematic representation of the mammalian cell cycle

(Nasmyth, 1996).
 24

1-1-Cell cycle and cancer:

Superficially, the connection between the cell cycle and cancer is

obvious: cell cycle machinery controls cell proliferation, and cancer is a
disease of inappropriate cell proliferation. Fundamentally, all cancers permit
the existence of too many cells. However, this cell number excess is linked in
a vicious cycle with a reduction in sensitivity to signals that normally tell a
cell to adhere, differentiate, or die. This combination of altered properties
increases the difficulty of deciphering which changes are primarily
responsible for causing cancer (Jacks and Weinberg, 1996).

The first genetic alterations shown to contribute to cancer

development were gain-of-function mutations. These mutations define a set
of "oncogenes" that are mutant versions of normal cellular "protooncogenes."
The products of protooncogenes function in signal transduction pathways that
promote cell proliferation. However, transformation by individual oncogenes
can be redundant (mutation of one of several genes will lead to
transformation) or can be cell type-specific (mutations will transform some
cells but have no effect on others). This suggests that multiple, distinct
pathways of genetic alteration lead to cancer, but that not all pathways have
the same role in each cell type (White, 1996).

More recently, the significance of loss-of-function mutations in

carcinogenesis has become increasingly apparent. Mutations in these so-
called "tumor suppressor" genes were initially recognized to have a major
role in inherited cancer susceptibility. Because inactivation of both copies of
a tumor suppressor gene is required for loss of function, individuals
heterozygous for mutations at the locus are phenotypically normal. Thus,
 25

unlike gain-of-function mutations, loss-of-function tumor suppressor

mutations can be carried in the gene pool with no direct deleterious
consequence. However, individuals heterozygous for tumor suppressor
mutations are more likely to develop cancer, because only one mutational
event is required to prevent synthesis of any functional gene product
(Morgenbesser et al., 1994).

It now appears that tumor suppressor gene mutations are highly

likely to promote, and may even be required for, a large number of
spontaneous as well as hereditary forms of cancer. Loss of function of the
tumor suppressor gene product pRb, for example, would be predicted to
liberate E2F transcriptional activators without requiring phosphorylation and
thus bypass a normal negative regulation controlling entry into the cycle.
Loss of the tumor suppressor gene product p16 would have a similar
consequence, liberating E2Fs by increasing pRb phosphorylation . In addition,
cell cycle progression can be halted at several points by the tumor suppressor
gene product p53, activated in response to checkpoints sensing DNA and
possibly also chromosome damage; loss of p53 would remove this brake to
cycling (Symonds et al., 1994).

2-Apoptosis and its markers:

Apoptosis and necrosis are too distinct, mutually exclusive, modes
of cell death. Apoptosis, frequently referred to as programmed cell death is
an active and physiological mode of cell death, in which the cell itself
designs and executes the program of its own demise and subsequent body
disposal. Different patterns of apoptosis (early and delayed apoptosis) many
cell types, cells of hematopoietic origin in particular, undergo apoptosis
 26

rapidly, to within few hours following exposure to relatively high

concentration of cytotoxic agents (Majino and Joris, 1995).

Apoptosis can be defined as 'gene-directed cellular self-destruction''

although this is really a phenomenon where cells are programmed to die at a
particular point, e.g. during embryonic development, and even here cells may
go through an apoptotic pathway.  However, apoptosis is certainly a distinct
process from other forms of oncosis leading to necrosis (Gerbaulet et al.,
2005 and Wallach et al., 1999).

Apoptosis affects individual cells, physiological induction e.g. lack

of signals, phagocytosis by macrophages or other cells and there is no
inflammatory response. Necrosis affects group of cells, non physiological
induction e.g. virus and poison, phagocytosis of macrophages and there is
inflammatory response (Wirth et al., 2005).

There are three different mechanisms by which a cell commits

suicide by apoptosis. In the intrinsic or mitochondarial pathway, the outer
membranes of mitochondria in a healthy cell express the protein; Bcl2 on
their surface. Bcl2 is bound to a molecule of the protein Apaf-1. Internal
damage to the cell (e.g., from reactive oxygen species) causes Bcl2 to release
Apaf-1; a related protein, Bax, to penetrate mitochondrial membranes
causing cytochrome c to leak out. The released cytochrome c and Apaf-1
bind to molecules of caspase 9 Fig. (2-2). The resulting complex of
cytochrome c, Apaf-1, caspase 9 and ATP is called the apoptosome. The
apoptosome aggregate in the cytosol (Niu et al., 2005 and Lam et al., 2005
and Kroemer& Reed 2000).
 27

Figure (2-2): The intrinsic or mitochondrial pathway (Lam et al., 2005).

 28

Caspase 9 is one of a family of over a dozen caspases. They are all

proteases. They get their name because they cleave proteins-mostly each
other at aspiratic acid residues. Caspase 9 cleaves and, in so doing, activates
other caspases. The sequential activation of one caspase by another creates an
expanding cascade of proteolytic activity (rather like that in blood clotting
and complement activation) which leads to digestion of structural proteins in
the cytoplasm, degradation of chromosomal DNA and phagocytosis of the
cell (Wada et al., 2005).

In the extrinsic or death receptor pathway, Fas and the TNF receptor
are integral membrane proteins with their receptor domains exposed at the
surface of the cell. Binding of the complementary death activator (FasL and
TNF respectively) transmits a signal to the cytoplasm that leads to activation
of caspase 8. Caspase 8 (like caspase 9) initiates a cascade of caspase
activation leading to phagocytosis of the cell Fig. (2-3). For example,
cytotoxic T cells recognize (bind to) their target, they produce more FasL at
their surface, this binds with the Fas on the surface of the target cell leading
to its death by apoptosis. In some cases, final destruction of the cell is
guaranted only withits engulfment by a phagocyte (Bijangi et al., 2005 and
Vega et al., 2005).

In the third way, neurons, and perhaps other cells, have another way
to self-destruct that unlike the two paths described above, doesn't use
caspase. Apoptosis- inducing factor (AIF) is a protein that is normally located
in the inter membrane space of mitochondaria. When the cell receives a
signal telling it that it is time to die, AIF is released from the mitochondrial, it
is migrates into the nucleus and binds to DNA, Which triggers the destruction
of the DNA and cell death (Urbano et al., 2005).
 29

Figure (2-3): The mechanism of apoptosis (Apoptosis triggered by

external signals: the extrinsic or death receptor path way) (Bijangi et al.,
2005).
 30

Defects in programmed cell death (apoptosis) mechanisms play

important roles in the pathogenesis and progression of hematological
malignancies, allowing neoplastic cells to survive beyond their normally
intended life-spans and subverting the need for exogenous survival factors.
Apoptosis defects also serve as an important complement to proto-oncogene
activation, as many deregulated oncoproteins that drive cell division also
trigger apoptosis (Evan and Littlewood, 1998).

Similarly, errors in DNA repair and chromosome segregation

normally trigger cell suicide as a defense mechanism for eradicating
genetically unstable cells, and thus apoptosis defects permit survival of the
genetically unstable cells, providing opportunities for selection of
progressively aggressive clones (Ionov et al., 2000).

Chemotherapy and irradiation trigger apoptosis in tumor cells and an

understanding of the biochemical pathways involved in apoptosis provides an
opportunity to classify tumors based on their response to common induction
regimens. Multiple distinct signaling pathways regulate apoptosis, but two
major cell death pathways have been implicated in hematological
malignancies: the mitochondrial pathway and the death receptor pathway Fig.
(2-4) (Evans et al., 2002).
 31

Figure (2-4): Diagram of the mitochondrial and death receptor pathways

of cell death (Evans et al., 2002).
 32

Both of these pathways ultimately activate members of the caspase

family of proteins that are responsible for executing the terminal phases of
apoptosis. p53 protein levels rise in response to various cellular stresses
including chemotherapy. p53 induces the loss of mitochondrial membrane
potential with subsequent release of cytochrome c, which forms a complex,
the "apoptosome," with the adapter molecule Apaf-1, ATP, and caspase-9.
This complex, in turn, activates caspase-3 (Evans et al., 2002).

Another proximal pathway of cell death involves death receptor

signaling at the cell surface. Binding of CD95-L and other tumor necrosis
factor (TNF) family ligands to their death inducing receptors, CD95/APO-
1/FAS or TNF- and TRAIL respectively, leads to receptor trimerization and
the recruitment of adapter molecules. These molecules include
FADD/MORT-1 that in turn lead to recruitment and activation of caspase-8.
This initiator caspase also cleaves and activates downstream caspases,
including caspase-3. Although generally described as being distinct, these
two proximal pathways are interconnected. For example, caspase-8 cleaves
the pro-apoptotic protein BID, which results in translocation to the
mitochondria and release of cytochrome c (Kishi et al., 2003, Blom, 2000, de
Franchis et al., 2000 and Goto et al., 2001).

Several studies have examined the prognostic significance of

apoptotic protein expression in leukemia. Defects in the p53 pathway are
distinctly rare in childhood malignancies including ALL, where mutations are
detected in < 5% of cases at the time of initial diagnosis. However, relapsed
blasts may harbor mutations of p53 gene much more commonly. Further,
ALL blasts at relapse have been noted to express high levels of the Mdm-2
protein, which abrogates p53 signaling (Dirven et al., 1995 and Pemble et
al., 1994).
 33

Cancer-associated defects in apoptosis play a role in chemoresistance

and radioresistance, increasing the threshold for cell death, and thereby
requiring higher doses for tumor killing (Tschopp et al., 1999 and Makin et
al., 2000).

Melanoma (skin cancer) cells avoid apoptosis by inhibiting the

expression of the gene encoding Apaf-1. Some cancer cells, especially lung
and colon cancer cells, secrete elevated levels of a soluble (decoy) molecule
that binds to FasL, plugging it up so it cannot bind Fas. Thus cytotoxic T
cells (CTL) cannot kill the cancer cells by the mechanism of death receptor
pathway. Other cancer cells express high levels of FasL, and can kill any
cytotoxic T cells (CTL) that try to kill them because CTL also express Fas
(but are protected from their own FasL) (Meijer et al., 2005).

Apoptosis plays a role in many diseases, such as cancer, viral

infections, and autoimmune and neurodegenerative disorders. There is a great
potential for treatment of these diseases in developing agents that can alter
the apoptotic process and change the natural disease progression. Molecules
whose roles in apoptosis have been investigated include Bcl-2 and c-myc
proteins, the p53 tumor suppressor gene and various tumor suppressor gene
products, MAP kinases, and proteases (Menendez et al., 2004).

2-1-The apoptosis promoter (p53):

p53 stimulates a wide network of signals that act through two major
apoptotic pathways. The extrinsic, death receptor pathway triggers the
activation of a caspase cascade, and the intrinsic, mitochondrial pathway
shifts the balance in the Bcl-2 family towards the pro-apoptotic members,
promoting the formation of the apoptosome, and consequently caspase-
 34

mediated apoptosis. The impact of these two apoptotic pathways may be

enhanced when they converge through Bid, which is a p53 target. The
majority of these apoptotic effects are mediated through the induction of
specific apoptotic target genes. However, p53 can also promote apoptosis by
a transcription-independent mechanism under certain conditions. Thus, a
multitude of mechanisms are employed by p53 to ensure efficient induction
of apoptosis in a stage-, tissue- and stress-signal-specific manner (Linda &
Carol, 1996 and Susan et al., 2003).

Some cancer causing viruses use tricks to prevent apoptosis of the

cells they have transformed. Several human papilloma viruses (HPV) have
been implicated in causing cervical cancer. One of them produces a protein
(E6) that binds and inactivates the apoptosis promoter p53. Binding of Fas
ligand or agonistic anti-Fas antibody to the death receptor Fas can activate a
caspase-cascade resulting in apoptosis. Fas cell surface expression was
determined by flow cytometry (Hougardy et al., 2005).

Genes involved in apoptosis are either pro-apoptotic (promote

apoptosis) or anti-apoptotic (inhibit apoptosis). P53 is a pro-apoptotic genes
present in all cells, but has special significance to cancer cells. It is a tumor
repressor gene, meaning that its presence reduces the occurrence of cancer
tumors by promoting apoptosis in cancer cells. Normally it induces apoptosis
by activating caspases 9, 8, 7, and 3. The loss of p53 decreases caspase
activation and therefore the cell will not undergo apoptosis. Mutation in the
p53 gene is the most common mutation in cancer; it is present in about half
of all cancer tumors, 80% in all colon cancer tumors, 50% of lung cancer
tumors, and 40% of breast cancer tumors (Polyak et al., 1997).
 35

Under normal conditions p53 is a short-lived protein. The p53

inhibitor Mdm2 (Hdm2 in humans) is largely responsible for keeping p53 in
this state. Mdm2 inhibits the transcriptional activity of p53 and, more
importantly, promotes its degradation by the proteasome (Levine, 1997).

p53 mutants in tumours have a reduced affinity for DNA and a

reduced ability to induce apoptosis. We describe a mutant with the opposite
phenotype, an increased affinity for some p53-binding sites and an increased
ability to induce apoptosis. The apoptotic function requires transcription
activation by p53 (Elisabeth et al., 1999).

Early observations suggested that p53 may function as an oncogene,

because overexpression of p53 appeared to cause oncogenic transformation
of cells. In the late 1980s, however, several critical discoveries defined the
normal function of p53 to be anti-oncogenic. Wild-type p53 genes, when
introduced into cells, were found to be growth suppressive (Isabela et al.,
2000).

p53 plays multiple roles in cells. Expression of high levels of wild-

type (but not mutant) p53 has two outcomes: cell cycle arrest or apoptosis. In
response to genotoxic stress, p53 acts as an "emergency brake" inducing
either arrest or apoptosis, protecting the genome from accumulating excess
mutations. Consistent with this notion, cells lacking p53 were shown to be
genetically unstable and thus more prone to tumors (Isabela et al., 2000).

p53 promotes cytochrome c release through the induction of target

genes encoding BH3-only proteins. Importantly, p53 also induces APAF-1
expression through a response element within the APAF-1 promoter (Kannan
et al., 2001)
 36

The tumor suppressor gene product p53 is clearly a central player in

many biochemical pathways that are pivotal to human carcinogenesis. The
sequence-specific DNA binding properties of this nuclear phosphoprotein
regulate the transcription of a continually expanding number of genes, the
protein products of which regulate cell cycle progression and apoptosis
(Isabela et al., 2000).

Loss of p53 function by mutation is common in cancer. However,

most natural p53 mutations occur at a late stage in tumor development, and
many clinically detectable cancers have reduced p53 expression but no p53
mutations. It remains to be fully determined what mechanisms disable p53
during malignant initiation and in cancers without mutations that directly
affect p53. p53 mutants in tumours have a reduced affinity for DNA and a
reduced ability to induce apoptosis (Niu et al., 2005).

p53 expression has important clinical implications as an indicator of

prognosis and response to chemotherapy or radiotherapy in different human
tumor types. The common effect of p53 mutations found in tumours is to
inactivate p53 as a transcription factor. Consequently, a great deal of effort
has been expended in trying to identify transcriptional targets of p53.
Particular attention has been paid to target genes which may mediate cell-
cycle arrest and apoptosis (Ko and Prives, 1996 ).

p53 dependent G1 and G2 arrest requires induction of the p21 cyclin-

dependent kinase inhibitor. In contrast, no single gene can explain p53-
induced apoptosis. Many p53 target genes have been identified which
function in known apoptotic pathways, regulate survival factor signalling,
induce apoptosis when over expressed or are involved in biochemical events
linked to apoptosis (Buckbinder et al., 1997 , Miyashita and Reed, 1995 ,
 37

Owen-Schaub et al., 1995, Polyak et al., 1997, Varmeh-Ziaie et al., 1997,

McCurrach et al., 1997 and Rampino et al., 1997).

p53 can activate target genes through a non-canonical sequence. The

first such example is in the p53-induced gene 3 (PIG3), which has been
implicated in the accumulation of reactive oxygen species and apoptosis
induction (Polyak et al., 1997). Another recently described example is the
gene encoding the pro-apoptotic phosphatase PAC1, which is induced
through binding of p53 to a novel palindromic binding site (Yin et al., 2003).

A novel insight into the interplay between p53 and its family
members, p63 and p73, in the induction of apoptosis has been recently
revealed (Flores et al., 2002). The effect of p63 and p73 on p53
transcriptional activity, using a selection of knockout mouse embryo
fibroblasts (MEFs), defined two distinct classes of target gene. Whereas p53
alone is sufficient for the induction of p21 and Mdm2, the induction of the
apoptotic genes PERP, Bax and Noxa requires p53 together with p63 and p73.
This finding demonstrates an essential role for both p63 and p73 in the
efficient induction of apoptotic target genes by p53. The mechanism of this
cooperation is currently unknown, but it may involve an enhanced binding to
and/or stabilization of the transcription complex on the promoters of p53
apoptotic target genes by the cooperative action of all three members (Urist
and Prives, 2002).

In addition to the contribution of p63 and p73 to the apoptotic

function of p53, they play an important role in the precise control of cell
death during normal mouse development. p73 also plays a role in the
induction of cell death in response to DNA damage, a process involving
cooperation between the Abl tyrosine kinase and p73 (Shaul, 2000).
 38

Immunohistochemical (IHC) detection of p53 expression has been

established as a relatively easy and straightforward method for fresh and
archival tissues. Available monoclonal antibodies recognize both wild-type
and mutant forms, but there may be a selective detection of the latter owing to
the very short half-life of the former (Porter et al., 1992 and Soussi et al.,
1994).

p53 is a tumor suppressor that is rarely mutated in ALL patients

but whose function is frequently altered by mutations to genes that code for
proteins that regulate p53 function. Activation of p53 occurs in response to
cells that have acquired DNA damage that may be engaged in aberrant cell
proliferation. Mutations to proteins that regulate p53 function, like HDM2,
p14, and p21, are frequent findings in ALL (Roman et al., 2002).

Bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast

cell lines were analyzed via flow cytometry (FCM) for expression of p53
protein along with their DNA content. Significantly elevated levels of the p53
protein was present in some but not all of the transformed cell lines.
Quantitation of p53 protein in cell lines containing BPV-1 DNA revealed that
the tumorigenic cell lines expressed higher levels of the p53 protein
(Agrawal et al., 1994).

The correlation between p53 abnormalities and DNA aneuploidy and

that analysis of p53 protein is useful for prediction of clinical course in
esophageal squamous cell carcinoma (Goukon et al., 1994).

Liu et al., (2004) evaluated changes in apoptotic proteins expression

that occur in response to chemotherapy in pediatric cases with acute leukemia
just prior to and 1, 6 and 24 hours following the administration of multiagent
 39

chemotherapy. They found great heterogeneity in the patterns of apoptotic

protein expression in the initial response to chemotherapy among individual
patient samples. Importantly, no increases in p53, p21 or Mdm-2 protein
expression were seen in leukemic blasts from the standard risk patients whose
initial treatment consisted of the non-p53-dependent drugs, vincristine and
prednisone.

In the subgroup of children who received at least one p53 dependent

drug, patients could be segregated into two groups, one group that showed
up-regulation of p53 protein and its target p21, and another group that
showed no increase following therapy, thus identifying at least two distinct
pathways leading to apoptosis (Chen et al., 1996).

2-2-Bcl2 proteins

Members of the Bcl-2 protein family play pivotal roles in the

decision and execution phases of apoptosis in the mitochondrial pathway. To
date, 24 Bcl-2 family members have been identified as either pro- (e.g., Bax,
Bak, Bcl-XS, Bid, Bad, and Noxa) or anti- (e.g., Bcl-2 and Bcl-XL) apoptotic
proteins. Bcl-2 proteins form homo- and heterodimeric complexes to regulate
mitochondrial channel formation and subsequent release of cytochrome c
from the mitochondria (Kishi et al., 2003, Blom, 2000, de Franchis et al.,
2000, Goto et al., 2001 and Cryns et al., 1999).

The Bcl2 family proteins are the central regulators of the

mitochondrial pathway. Bcl2 is an inhibitor of apoptosis. Bcl2 and its human
homolog introduce a new category of oncogenes that act by decreasing cell
death. Over expression of Bcl2 promotes oncogensis by repressing cell death
 40

and extending cell life. However, overexpression can also lead to retardation
of cell cycling via prolongation of the G1 phase of the cycle (Webb et al.,
2005 and Green & Reed, 1998).

The Bcl2 family of intracellular proteins is the central regulator of

caspase activation, and its opposing factions of anti- and pro-apoptotic
members arbitrate the life-or-death decision. The oncogenic activity of the
Bcl2 gene is carried out via suppression of lymphocytic apoptosis or
programmed cell death. (Cory & Adams, 2002 and Roumier et al., 2002).

BCL2 is an important regulator of apoptosis, first identified from its

involvement in follicular B cell lymphoma, where the common t(8:14)
translocation causes the activation of the BCL2 oncogene. BCL2 is now
recognised as a survival factor for many types of cell, notably neurons.
Expression of BCL2 is widespread during embryogenesis but is restricted to
long-lived cells in the adult. A critical mediator of BCL2 apoptosis is
interleukin-1 beta-converting enzyme (ICE) a cysteine protease that
processes IL-1 beta during the inflammatory response (Roumier et al., 2002).

BCL2 is a member of a multigene family (highly conserved

evolutionarily with viral homologues). Other proteins in the family (BCLX,
BAD, BAX, BAD etc) antagonise inhibition of apoptosis by binding to
BCL2. Hence the balance of various members of the BCL family determines
the extent to which cell death is promoted or prevented. This model is
consistent with the findings of high levels of BCL2 in a variety of solid
tumours (Jiang and Milner, 2003).

Apoptosis can also be induced by a variety of cytokines e.g. TGF

beta family, which inhibit the proliferation of a wide variety of cell types that
 41

may undergo concomitant cell death. TGF beta induced apoptosis is blocked
in myeloblastic leukaemia cells by BCL2 expressed at a level that does not
block but merely delays p53-induced apoptosis. This may reflect the fact that
both TGF beta and p53 suppress BCL2 but only p53 has the ability to
activate BAX, thus deflecting the expression pattern towards apoptosis
(Seckin et al., 2002).

Active cell suicide (apoptosis) is induced by events such as growth

factor withdrawal and toxins. It is controlled by regulators, which have either
an inhibitory effect on programmed cell death (anti-apoptotic) or block the
protective effect of inhibitors (pro-apoptotic). Many viruses have found a
way of countering defensive apoptosis by encoding their own anti-apoptosis
genes preventing their target-cells from dying too soon. All proteins
belonging to the Bcl-2 family contain either a BH1, BH2, BH3, or BH4
domain. All anti-apoptotic proteins contain BH1 and BH2 domains, some of
them contain an additional N-terminal BH4 domain (Bcl-2, Bcl-x (L), Bcl-
w), which is never seen in pro-apoptotic proteins, except for Bcl-x(S). On the
other hand, all pro-apoptotic proteins contain a BH3 domain (except for Bad)
necessary for dimerization with other proteins of Bcl-2 family and crucial for
their killing activity, some of them also contain BH1 and BH2 domains (Bax,
Bak). The BH3 domain is also present in some anti-apoptotic protein, such as
Bcl-2 or Bcl-x (L). Proteins that are known to contain these domains include
vertebrate Bcl-2 (alpha and beta isoforms) and Bcl-x (isoforms (Bcl-x(L) and
Bcl-x(S)) (Poliseno et al., 2002).

Antiapoptotic B cell leukemia/lymphoma (BCL2) family

proteins are expressed in many cancers, but the circumstances under which
these proteins are necessary for tumor maintenance are poorly understood.
A novel functional assay that uses Bcl2 homology domain (BH3) peptides
 42

to predict dependence on antiapoptotic proteins was exploiteded , a strategy,

BH3 profiling. BH3 profiling accurately predicts sensitivity to Bcl2
antagonist ABT-737 in primary chronic lymphocytic leukemia (CLL) cells.
BH3 profiling also accurately distinguishes myeloid cell leukemia sequence
1 (MCL1) from Bcl2 dependence in myeloma cell lines. It was shown that
the special sensitivity of CLL cells to Bcl2 antagonism arises from the
requirement that Bcl2 tonically sequester proapoptotic BIM in CLL. ABT-
737 displaced BIM from Bcl2's BH3-binding pocket, allowing BIM to
activate BAX, induce mitochondrial permeabilization, and rapidly commit
the CLL cell to death. It was demonstrated that Bcl2 expression alone does
not dictate sensitivity to ABT-737. Instead, Bcl2 complexed to BIM is the
critical target for ABT-737 in CLL. An important implication is that in
cancer, Bcl2 may not effectively buffer chemotherapy death signals if it is
already sequestering proapoptotic BH3-only proteins. Indeed, activator
BH3-only occupation of Bcl2 may prime cancer cells for death, offering a
potential explanation for the marked chemosensitivity of certain cancers that
express abundant Bcl2, such as CLL and follicular lymphoma (Del Gazio et
al., 2007).

The relationship between gene expression of Bcl 2 and Bax and the
therapeutic effect in oral cancer patients had investigated. A significant
correlation between Bcl-2/Bax gene expression ratio in the peripheral blood
mononuclear cells (PBMCs) from the patients, and the therapeutic effect of
radiation therapy These findings suggested that Bcl-2 and Bax gene
expression in PBMCs may be useful as a prognostic factor in oral cancer
patients (Oshikawa et al., 2006).

Epstein-Barr virus (EBV), the cause of mononucleosis and cause of

Burkitt's lymphoma produces a protein similar to Bcl2 and produces another
 43

protein that causes the cell to increase its own production of Bcl2. Both these
actions make the cell more resistant to apoptosis (thus enabling the cancer
cell to continue to proliferate). Even cancer cells produced without the
participation of viruses may have tricks to avoid apoptosis (Lu et al., 2005).

Some B-cell leukemias and lymphomas express high levels of Bcl2,

thus blocking apoptotic signals they may receive. The high levels result from
a translocation of the Bcl2 gene into an inhancer region for antibody
production (Menendez et al., 2004).

Bcl2-L12 contributes to the classical tumor biological features

of Glioblastoma (GBM) such as intense apoptosis resistance and florid
necrosis, and may provide a target for enhanced therapeutic responsiveness
of this lethal cancer (Stegh et al., 2007).

2-3C-myc oncogene:
The c-myc gene was discovered as the cellular homolog of the retro
viral v-myc oncogene 20 years ago. The c-myc proto-oncogene was
subsequently found to be activated in various animal and human tumors. It
belongs to the family of myc genes that includes B-myc, L-myc, N-myc, and
s-myc; however, only c-myc, L-myc, and N-myc have neoplastic potential
(Wechsler et al., 1997 and Facchini & Penn, 1998). Targeted homozygous
deletion of the murine c-myc gene results in embryonic lethality, suggesting
that it is critical for development. Homozygous inactivation of c-myc in rat
fibroblasts caused a marked prolongation of cell doubling time, further
suggesting a central role for c-myc in regulating cell proliferation (Mateyak
et al., 1997).
 44

Bovine papillomavirus type 1 (BPV-1)-transformed mouse

fibroblast cell lines were analyzed via flow cytometry (FCM) for expression
of c-myc protein along with their DNA content. Significantly elevated levels
of the c-myc protein was present in some but not all of the transformed cell
lines. Quantitation of c-myc protein in cell lines containing BPV-1 DNA
revealed that the tumorigenic cell lines expressed higher levels of the c-myc
protein (Agrawal et al., 1994).

The role of c-Myc in the cell cycle has been a confusing area due to
the collection of data from different experimental models, although it is well
established that c-myc is an early serum response gene. It should be noted
that models of serum or growth factor stimulation of starved cells primarily
address the G0/G1 and G1/S transitions. Therefore, early studies implicated c-
Myc in the G0/G1 transition. In cycling cells, however, the participation of c-
Myc in the cell cycle may be different. Furthermore, in anchorage-dependent
cell growth, c-Myc may affect other components of the cell cycle (Amati et
al., 1998).

It is proposed that c-Myc induces apoptosis through separate 'death

priming' and 'death triggering' mechanisms in which 'death priming' and
mitogenic signals are coordinated. Investigation of the mechanisms that
underlie the triggering steps may offer new therapeutic opportunities
(Prendergast, 1999).

The antiapoptotic effect of Epstein-Barr virus (EBV) was associated

with a higher level of Bcl-2 expression and an EBV-dependent decrease in
steady-state levels of c-MYC protein. Although the EBV EBNA-1 protein is
expressed in all EBV-associated tumors and is reported to have oncogenic
potential, enforced expression of EBNA-1 alone in EBV-negative Akata cells
 45

failed to restore tumorigenicity or EBV-dependent down-regulation of c-

MYC. These data provide direct evidence that EBV contributes to the
tumorigenic potential of Burkitt lymphoma and suggest a novel model
whereby a restricted latency program of EBV promotes B-cell survival, and
thus virus persistence within an immune host, by selectively targeting the
expression of c-MYC (Ingrid et al., 1999).

Much recent research on c-Myc has focused on how it drives

apoptosis. c-Myc is widely known as a crucial regulator of cell proliferation
in normal and neoplastic cells, but until relatively recently its apoptotic
properties, which appear to be intrinsic, were not fully appreciated. Its death-
dealing aspects have gained wide attention in part because of their potential
therapeutic utility in advanced malignancy, where c-Myc is frequently
deregulated and where novel modalities are badly needed. Although its exact
function remains obscure, c-Myc is a transcription factor and advances have
been made in characterizing target genes which may mediate its apoptotic
properties (Hermeking, 2003).

Ectopic expression of c-Myc (Myc) in most primary cell types

results in programmed cell death, and malignant transformation cannot occur
without additional mutations that block apoptosis. The development of Myc-
induced lymphoid tumors was studied. Myc can be upregulated in acute
myeloid leukemia (AML), but its exact role in myeloid leukemogenesis is
unclear. To study its role in AML, a murine stem cell virus (MSCV)
retroviral gene transfer/transplantation system was used to broadly express
Myc in the bone marrow of mice either alone or in combination with
antiapoptotic mutations. Myc expression in the context either of Arf/Ink4a
loss or Bcl-2 coexpression induced a mixture of acute myeloid and acute
lymphoid leukemias (AML+ALL). In the absence of antiapoptotic mutations
 46

however, all mice transplanted with MSCV-Myc developed AML exclusively.

MSCV-Myc-induced AML was polyclonal, readily transplantable, possessed
an intact Arf-p53 pathway, and did not display cytogenetic abnormalities by
spectral karyotyping analysis. Lastly, it was found that Myc preferentially
stimulated the growth of myeloid progenitor cells in methylcellulose. These
data provided the first direct evidence that Myc is a critical downstream
effector of myeloid leukemogenesis and suggested that myeloid progenitors
are intrinsically resistant to Myc-induced (Hui et al., 2005).
 47

III-Flow cytometry

Flow cytometry is a laser-based technology that is used to measure

characteristics of biological particles. This technology is used to perform
measurements on whole cells as well as prepared cellular constituents such as
nuclei and organelles (Melamed et al., 1990, Tileney et al., 1996 and
McCoy, 2002).

The flow cytometer is an instrument for measuring scattered and

fluorescent light from single particles. The physics of the interaction of light
with single particles provides the scientific foundation for the design and
operation of the flow cytometer and for the critical evaluation of flow
cytometric data (Scornik et al., 1994).

Flow cytometry uses the principles of light scattering, light excitation,

and emission of fluorochrome molecules to generate specific multi-parameter
data from particles and cells in the size range of 0.5um to 40um diameter.
Cells are hydro-dynamically focused in a sheath of phosphate buffer saline
(PBS) before intercepting an optimally focused light source. Lasers are most
often used as a light source in flow cytometry (Talbot, 1993).

The technology of flow cytometry and the discovery of a method to

produce monoclonal antibodies have made possible the clinical use of flow
cytometry for the identification of cell populations. Light scatter is utilized to
identify the cell populations of interest, while the measurement of
fluorescence intensity provides specific information about individual cells.
Monoclonal antibodies (tagged) with the fluorescent dye are commonly used
for the identification of cell surface antigens and fluorescent dyes that
 48

directly and specifically bind to certain components of the cell (i.e. DNA) are
used for cell cycle analysis (Reckenwald, 1993 and Shapiro, 1995).

Cells or particles are prepared as single-cell suspension for flow

cytometric analysis. This allows them to flow single file in a liquid stream
past a laser beam. As the laser strikes the individual cells. First light
scattering occurs that is directly related to structural and morphological cell
features. Second, fluorescence occurs if the cells are attached to a fluorescent
probe. Fluoresent probes are typically monoclonal antibodies that have been
conjucated to fluorochromes; they can also be fluorescent stains reagents that
are not conjugated to antibodies (Parks and Herzenberg, 1989 and
Rechenwald et al, 1993).

Fluorescent probes are reacted with the cells or particles of interest

before analysis; therefore, the amount of fluorescence emitted as a particle
passes the light source is proportional to the amount of fluorescent probe
bound to the cell or cellular constituent (Radcliff and Jaroszeski, 1998).

After acquisition of light scattering and fluorescence data for each

particle, the resulting information can be analyzed utilizing a computer and
specific software that are associated with the cytometer (Rose et al,. 1992
and Longobardi-Given, 1992).

There are two distinct types of flow cytometers that can be used to
acquire data from particles. One type can perform acquisition of light
scattering and fluorescence only. The other type is capable of acquiring
scattering and fluorescence data but also has the powerful ability to sort
particles. Both types function in a similar manner during acquisition, for
example FACScan (Becton Dickinson), this equipped with an air –cooled 15
 49

mw argon ion laser emitting at 488 nm. Three fluorescence channels can be
measured as well as two light scatter parameters. The FACScan is also
equipped with a doublet discrimination module allowing the analysis of the
cell cycle. The FACScan is user-operated (after instruction) and is available
for use 24 hours per day (Kandathil et al., 2005).

However, sorting instruments have the powerful ability to physically

separate particles based on light scattering and/or fluorescent emission
characteristics. Cytometers were originally designed to sort, for example
FACS caliber 1, 2 (Becton Dickison), this used for analysis only. Unlike the
FACScan which is a dual laser system. The primary laser is an air-cooled 15
mw argon ion laser emitting at 488 nm thus allowing two light scatter
parameters and three fluorescence channels to be measured. The second laser
is ared diode laser emitting at 635nm. Thus allowing for the excitation of
other dyes such as allophycocyanin or to-pro-3 with power Macintosh G4
running system 9.0 and cell Quest v 3.3. Thus, cytometers that perform
acquisition without sorting are the most common of the two types (Rose et
al., 1992).

1-Principles of flow cytometric instrumentation:

Flow cytometers are very complex instruments that are composed

of four closely related systems. The fluidic system transports particles from
a suspension through the cytometer for interrogation by an illumination
system. The resulting light scattering and fluorescence is collected, filtered,
and converted into electrical signals by the optical and electronics system.
The data storage and computer control system saves acquired data and is
also the user interface for controlling most instrument functions. These four
systems provide a very unique and powerful analytical tool for researchers
 50

and clinicians. This is because they analyze the properties of individual

particles, and thousands of particles can be analyzed in a matter of seconds.
Thus, data for a flow cytometric sample are a collection of many
measurements instead of a single bulk measurement (Radcliff and
Jarosezeski, 1998)..

Histograms are the simplest modes of data representation.

Histograms allow visualization of a single acquired parameter. Mean
fluorescence and distributional statistics can be obtained based on markers
that the user can graphically set on the plot. Multiple histograms can be
overlaid on one another to depict qualitative and quantitative differences in
two or more samples. Two-parameter data plots are somewhat more
complicated than histograms; however, they can yield more information.
Two-parameter dot plots of FSC vs SSC allow visualization of both light-
scattering parameters that are important for identifying populations of
interest. Bivariate fluorescent plots allow discrimination of dual-labeled
populations that might remain hidden if histograms were used to display
fluorescent data. Two-parameter plots that combine one light-scattering
parameter and a fluorescent parameter are useful for analyzing control
samples to elucidate the origin of nonspecific binding. Data analysis is very
graphically oriented. Experience and pattern recognition become important
when using two-parameter data plots for qualitative as well as quantitative
analysis. The technique of gating or drawing regions on dual parameter
light-scatter plots allows one to exclude information and examine the
population of interest by disallowing particles that might confound or
interfere with analysis. This is one of the fundamental uses for gating
(Radcliff and Jarosezeski, 1998).
 51

Flow cytometers can be described as four interrelated systems which

are shown in Fig. (3-1). these four basic systems are common to all
cytometers regardless of the instrument manufacturer and whether or not the
cytometer is designed for analysis or sorting (Melamed et al., 1990 and
Longobardi-Given, 1992 Owens & Loken, 1995).

1-1-Fluidic system:
The fluidic system is the heart of a flow cytometer and is responsible
for transporting cells or particles from a prepared sample through the
instrument for data acquision Fig. (3-2). The primary component of this
system is a flow chamber. The fluidic design of the instrument and the flow
chamber determine how the light from the illumination source ultimately
meets and interrogates particles. Typically, a diluent, such as phosphate
buffered saline, is directed by air pressure into the flow chamber. This fluid is
referred to as sheath fluid and passes through the flow chamber after which it
is intersected by the illumination source. Then, the sample under analysis, in
the form of a single particle suspension, is directed into the sheath fluid
stream prior to sample interrogations. The sample then travels by laminar
flow through the chamber (Ormerod, 1994).
 52

Figure (3-1): Facscaliblur flow cytometry instrument.

 53

Figure (3-2): Flow cytometer system (Facscalibur) (Ormerod, 1994).

 54

The pressure of the sheath fluid against the suspended particles

aligns the particles in a single file fashion. This process is called
hydrodynamic focusing and allows each cell to be interrogated by the
illumination source individually while traveling within the sheath fluid
stream (Radcliff and Jaroszeski, 1998).

The flow cell is the functional core of the fluidic system because it
presents cells in a single file for interrogation by the cytometer illumination
system. A typical flow cell Fig. (3-3) consists of a converging nozzle in
which sample is introduced at low flow rates into a larger laminar flow of
isotonic saline or sheath fluid. The cells in the sample follow the converging
streamlines and are hydrodynamically focused into alignment. The sample is
injected into the center of a sheath flow. The combined flow is reduced in
diameter, forcing the cell into the center of the stream. This the laser one cell
at a time. This schematic of the flow chamber in relation to the laser beam in
the sensing area (Philip, 2002).

1-2-Illumination system:
Flow cytometers use laser beams that intersect a cell or particle that
has been hydro dynamically focused by the fluidic system. Light from the
illumination source passes through a focusing apparatus before it intersects
the sample stream. This apparatus is a lens assembly that focuses the laser
emission into a beam with an elliptical cross-section that ensures a constant
amount of particle illumination despite any minor positional variations of
particles within the sample stream (Zimmermann and Truss, 1979).
 55

Laser options

Figure (3-3): Flow cytometers use the principle of hydrodynamics

focusing for presenting cells to a laser (Philip, 2002).
 56

Light and fluorescence are generated when the focused laser beam
strikes a particle within the sample stream. These light signals are then
quantitated by the optical and electronics system to yield data that is inter-
prêt able by the user (Shapiro, 1995).

Two systems are used in flow cytometry to focus the illuminating

light to the point at which it intersects the cell stream. One type of system
uses a spherical lens to give a focal spot size of 30- 60 µm. The second
system uses a pair of crossed cylindrical lenses to focus the light to a sheet
about 120 µm wide and 4-7 µm deep (Cledat et al., 2004).

Most flow cytometers utilize a single laser, however, some systems

support the simultaneous use of two or more different lasers. The most
commonly used laser is an argon ion laser that has been configured to emit
light in the visible range of the spectrum. A 488- nm. Laser emission is used
for most standard applications. The majority of fluorochrom that are
available on the market today can be excited using this wavelength. Thus,
laser is excellent excitation sources because they provide a single wavelength
beam that is also stable, bright, and narrow. Some type of lasers present in
flow cytometers can be turned to U.V. or other wavelengths. If the exiting is
not tunable, then another laser source that emits the desired wavelength is
required. At the measuring point in a typical flow cytometer the stream of
cells intersects a beam of light from a laser or arc lamp Fig. (3-4). When light
interacts with biological particles some of the light is scattered out of the
incident beam and this scattered light may be collected over a range of angles
by detectors positioned around the measuring point (Gerstner et al., 2005).
 57

Figure (3-4): A simplified illustration of Flow Cytometry (Gerstner et al.,

2005).
 58

1-3-Optical and electronics system:

The excitation optics consists of the light source and the optical
components that serve to interrogate or excite the hydrodynamically focused
sample stream in the flow cell. The 488 nm line of the argon laser is used as a
light source in many commercially available cytometers, but any light source
that provides the requisite intensity, e.g. , the mercury vapor or the xenon are
lamp can be used. Optical components are used to expand, shape and focus
the light which then interacts with the sample in the flow cell. The flow cell
is usually made of quartz and is designed to minimize diffraction and to
maximize the collection of the optical signals. The light source is often a
laser. Laser is used because they provide a very concentrated and intense
beam of monochromatic character of the light is especially important in
making fluorescence measurements (Telford, 2004).

The amount of light that is scattered by a cell is a complex function

of its size, shape and refractive index. The sensitivity of light scattering to
each of these factors is dependent upon the range of angles over which the
scattered light. The light scattered at small angles (i.e. forward light scatter)
could be successfully used to determine relative volume distributions for
populations of cells (Wang et al., 2004).

Light scattered and emitted in all directions (360º) after the laser
beam strikes an individual cell or particle that has been hydrodynamically
focused. The optical and electronics system of a typical flow cytometer is
responsible for collecting and quantitating at least five types of parameters
from this scatter light and emitted fluorescence. Two of these parameters are
light scattering properties. Light that is scattered in the forward direction (in
the same direction as the laser beam) is analyzed as one parameter, and light
scattered at 90º relative to the incident beam is collected as a second
 59

parameter. Forward-scattered (FSC) light is a result of diffraction. Diffracted

light provides basic morphological information such as relative cell size that
is referred to as forward angle light scatter (FSC). Light that is scattered at
90º to the incident beam is the result of refracted and reflected light. This
type of light scatters is referred to as side-angle light scatter (SSC). This
parameter is an indicator of granularity within the cytoplasm of cell as well
as surface membrane irregularities to topographies (Philip, 2002).

Most current laboratory bench-top flow cytometers are capable of

detecting fluorescence from three different regions of the visible spectrum.
Cutometers are optically conquered to detect a narrow range of wave lengths
in each region. Fluorescence emission is detected simultaneously along with
FSC and SSC data; therefore, up to five parameters can be simultaneously
measured for each analyzed sample (Longobardi- Given, 1992)

Fluorescence is detected using networks of mirrors, optics, and beam

splitters that direct the emitted fluorescent light toward highly specific optical
filters. The filters collect light within the range of wave lengths associated
with each of the three fluorescent channels. Filtered light is directed toward
photo multiplier tubes or PMTs for conversation into electrical signals
(Telford, 2004).

1-4-Data storage and computer control system:

After light scattering and fluorescence is converted to electrical
signals by the optical and electronics system, the information is converted
into digital data that the computer can interpret. The signals generated from
cells or particles are referred to as events and are stored by the computer
(Rose et al., 1992).
 60

After the different signals or pulses are amplified they are processed
by an Analog to Digital Converter (ADC) which in turn allows for events to
be plotted on a graphical scale (One Parameter, Two parameter Histograms).
Flow cytometry data outputs are stored in the form of computer files
(Radcliff and Jaroszeski, 1998).

Histogram files can be in the form of one-parameter or two-

parameter files. Histogram files consist of a list of the events corresponding
to the graphical display specified in your acquisition protocol. A one-
parameter histogram is a graph of cell count on the y-axis and the
measurement parameter on x-axis. All one-parameter histograms have 1,024
channels. These channels correspond to the original voltage generated by a
specific "light" event detected by the PMT detector. In other words, the ADC
assigns a channel number based on the pulse height for individual events.
Therefore, brighter specific fluorescence events will yield a higher pulse
height and thus a higher channel number when displayed as a histogram. A
graph representing two measurement parameters, on the x- and y-axes, and
cell count height on a density gradient. This is similar to a topographical
map. You can select 64 or 256 channels on each axis of two-parameter
histograms. Particle counts are shown by dot density or by contour plot. Fig.
(3-5) (Roederer et al., 2004).

List-mode files consist of a complete listing of all events

corresponding to all the parameters collected, as specified by your acquisition
Protocol. This file follows a format specified by the FCS 3.0 standard. Raw
list-mode data files can be opened or replayed using any program designed
for analysis of flow cytometry data. You should keep in mind that a Protocol
serves as a template. It allows you to collect specified Parameters (i.e. FLS,
FL1, FL2, etc.), and how these parameters are displayed. Protocols also serve
 61

to determine how the data is gated, and contains all the Regions from which
your statistics will be generated. In addition, Protocols contain other specific
information that serves as direct interface between the computer workstation
and the cytometer. These pertain to high voltage settings for the PMT
detectors, gains for amplification of linear parameters, sample flow rates,
fluorescence compensation, discrimination settings, etc. Once your data has
been collected and written into a list-mode file you can replay the file either
using the specific Protocol used for collection or any other program
specifically designed for analysis of flow cytometry data. However, you
should keep in mind that you can only adjust Regions, Gating, and
Parameters to be displayed. Settings such as amplification, fluorescence
compensation, etc., can not be modified. Therefore, when collecting data
make sure that your instrument settings are correct. Finally, if you open your
listmode files using a programs such as FlowJo Fig. (3-6), WINMIDI, and/or
ExPO you will have to specify parameter displays, and create Regions and
Gating corresponding to the Protocol used for collecting the data (McCoy,
2002).
 62

Figure (3-5): Two parameter histogram and dot plot displaying FL1-
FITC on the x axis and FL2-PE on the y axis (Roederer et al., 2004).
 63

Figure (3-6): FlowJo program (McCoy, 2002).

 64

The number of events acquired for each sample is always determined

before analysis and is usually set using software designed to control
cytometer operation. A conventional acquisition value is 100.000 events per
sample. However this value may vary and range upward of events per sample
depending on the experimental objective (Melamed et al., 1990).

2-Data analysis:

Data analysis is a very critical part of any experiment that utilizes

flow cytometry. Data is analyzed using a computer and software is usually
specific to flow cytometric data and is often part of the same computer
system that is used to control the instrument during acquision. The most
common display is a histogram. A typical histogram data plot is shown in
Fig. (3-7, 3-8) (Abu- Absi et al., 2003).

It is also possible to display two parameters simultaneously such as

FSC vs SSc or FL1 vs FL2. For two parameter plots, data from a population
of individual particles can be displayed in the form of dots or as contours
shown in Fig. (3-9) (Parks and Henzenberg, 1989).

Contour density plots display the data from a population of cells as a

series of concentric lines that correlate to different cell or particle densities
within the axes. Dot-plots are probably the most common type of two-
parameter plots, and they are also the easiest to understand (Robinson,
1993).
 65

Figure (3-7): Analysis pulse width versus pulse height or area we can
eliminate the majority of G0 doublets that appear as G2 (Abu- Absi et
al., 2003).
 66

Figure (3-8): DNA histogram (Abu- Absi et al., 2003).

 67

Figure (3-9): DNA histogram (aneuphliod population) (Parks and

Henzenberg, 1989).
 68

IV- Applications of flow cytometry

Flow cytometers are very complex instruments that are composed of

four closely related systems. They provide a very unique and powerful
analytical tool for researchers and clinicians. Therefore, the flow cytometer is
widely used in research as well as in clinical immunology and hematology to
perform rapid immunophenotyping, cell sorting, and DNA analysis
(Longobardi-Given, 1992 and Bogh & Duling, 2005).

Flow cytometry is used for immunophenotyping and DNA content of

a variety of specimens including whole blood, bone marrow, serous cavity
fluids, cerebrospinal fluid, urine and solid tissues. Characteristics that can be
measured include cell size, cytoplasmic complexity, DNA or RNA content,
and a wide range of membrane-bound and intracellular portents and sorting
the cells (Rechtnwald, 1993, Ormerod, 1994 and Assuncao et al., 2005).

The use of flow cytometry in the clinical laboratory has grown

substantially in the past decade. This is attributed in part to the development
of smaller user friendly, less expensive instruments and a continuous increase
in the number of clinical applications as shown in Table (4-1) (Brown and
Wittwer, 2000).

Flow cytometry provides rapid analysis of multiple characteristics of

single cells. The information obtained is both qualitative and quantitative.
Where as in the past flow cytometers were found only in larger academic
 69

centers, advances in technology now make it possible for community

hospitals to use this methodology (Orfao et al., 1995 and McCoy, 2002).

Table (4-1): Common clinical uses of flow cytometry (Brown and

Wittwer, 2000).

Common characteristic
Field Clinical application
measures

Immunology Histocompitability crossmatching IgM, IgG

Transplantation rejection CD3, CIRCULATING OKT3

HLA-B27 detection HLA-B27

Immunodeficiency studies CD4, CD8

Oncology DNA content and S phase of tumors DNA

Measurements of profilation markers Ki-67, PCNA

Hematology Leukemia and Lymphoma phenotyping Leukocyte surface antigens

Identification of prognostically important Tdt, MPO

subgroups

Hematopiotic progenit or cell enumeration CD34

Diagnosis of systematic mastocytosis CD25,CD69

Reticylocyte enumeration RNA

Autoimmuneand alloimmune disorders

Antiplatelats disorders IgG, IgM

Anti-neutrophils antibodies IgG

Immune complexes Complement, IgG

Feto-maternal hemorrge quantification Hemoglobin F, rhesus D

 70

Immunohematology Erthrocyte surface antigens

Assessment of leukocyte contamination of blood Forward AND Side scatter

products

Genetic
PNH CD55,CD59
disorders

Functions of cells can be defined through the application of

fluorochrome dyes that have an affinity for cellular components.
Traditionally, common clinical applications are immunophenotyping of cells
of the hematopoietic system with fluorescent-labeled antibodies raised
against specific cell surface proteins (Davis et al., 2002).

Other approaches have been used to elucidate changes in cell

function and DNA content. Examples of clinical applications in equine
patients include immune-mediated hemolytic anemia, immune-mediated
thrombocytopenia (IMT), chronic inflammatory disease, and neoplasia
(Davis et al., 2002).

The great advantage of flow cytometry is that its applications are

highly amenable to standardization. The efforts that have been made for flow
cytometric applications in four major fields of clinical cell analysis: CD4+ T-
cell enumeration, CD34+ hematopoietic stem and progenitor cell
 71

enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma

immunophenotyping (Keeney et al., 2004).

The diagnosis of many primary immunodeficiency diseases requires

the use of several laboratory tests. Flow cytometry is applicable in the initial
workup and subsequent management of several primary immunodeficiency
diseases (Illoh, 2004).

1- Cell cycle analysis:

The measurement of the DNA content of cells was one of the first
major applications of flow cytometry and is still one of the biggest
applications in this laboratory today (Albro et al., 1993).

Flow cytometry offers the possibility to study several parameters

simultaneously, e.g. cell cycle modulation, mode of cell death, activity of
mitochondria. The phases of the cell cycle were determined and the induction
of apoptosis and necrosis was demonstrated by annexin binding, analysis of
mitochondrial membrane potential and DNA strand breaks (Tusch and
Schwab, 2005).

DNA ploidy and proliferative activity (S-phase fraction) are the two
biological parameters commonly measured by DNA flow cytometric
analysis. The prime purpose of most studies is the investigation of the
prognostic value of DNA flow cytometry in addition to the information
provided by conventional clinicopathological factors known to affect disease
prognosis. The general statement, for tumors in the same histopathological
stage of the disease, is that diploid and/or low proliferative tumors have a
more favourable prognosis than aneuploid and/or high proliferative tumors,
 72

suggesting an important role of DNA flow cytometry in the assessment of

tumor behaviour and in the outcome evaluation of the disease. (Pinto et al.,
2002).

1-1-Staining procedure:
The preparation and staining of cell suspension are the major factors
determining the validity and reproducibility of flow cytometric analysis.
There is no flow cytometric staining procedures which is universally
accepted and a number of different protocols have been advocated. All of the
DNA specific stains and the phenanthridinium dyes have been used for total
DNA staining of chromosomes. The former group has the potential
disadvantage that UV excitation is required but this constitutes no problem
for mercury arc lamp based system or those with a laser tunable to UV lines
(Hartwell, 1998).

The DNA fluorochromes in current use were classified into groups.

Stains that intercalate with double stranded nucleic acid and include (PI),
(EB) and (AO);and DNA specific stains that have a particular specificity for
moieties in DNA and include mithramiycin (MMC), ethedium
bromide/mithramicin (EB/MMC), a bisbenzimidazole derivative and 4-6
diamino-2- phenylindole (DAPI) (Taylor and Mithorpe, 1980).

Propidium iodide is bound to polynucleotide both by means of

intercalation. This is only affected to a limited extent by high ionic strengths
and electrostatically to secondary binding sites. The binding contents of these
later sites are greatly depending on the ionic strength of the medium and can
be eliminated by using sufficiently high ionic strengths. And also have
optimal excitation at a 488 nm laser and produce good results with RNAse
treatment (Hartwell, 1998).
 73

1-2-Evaluation of DNA histogram:

The DNA histogram is a very simple data set which
characteristically contains two peaks separated by a trough Fig.
(4-1). The first peak, which is usually the larger, corresponds to cells with
G0/G1 DNA content and the second, which should be at double the
fluorescence intensity of the first, corresponds to cells with G2+M DNA
content. Any cell scored in the trough has a DNA content intermediate
between G1 and G2+M and these usually represent cells in S-phase. In a
perfect data set, which doesn't exist, all G1 and G2+M cells would be scored
in single channels and any cells between or immediately adjacent to these
would be in S- phase (Henderson, 1998).

Since the cell material always contained normal diploid cells such as
leukocytes or normal kidney cells, these were used as an internal standard
and regarded as diploid (2C) (Tribukati, 1984).

When in doubt, lymphocytes should be added to establish the diploid

DNA value. Human Ficol-prepared lymphocytes, fixed in ethanol, were used
as external standard. The magnitude of the signal was adjusted so as to have
the standard diploid peak in a certain channel. In necessary the illumination
was adjusted so that the coefficient of variation (CV) of the resulting
lymphocyte diploid peak was less than 3% Fig. (4-1). All cell population
with G1 maximum deviating less than 10% form the standard value were
regarded as diploid (Gustafson, 1982).

Only G0/G1 peak is observed in DNA diploid. A broad peak

described by a large coefficient of variation may obscure a second peak. The
coefficient of variation of the G0/G1 peak must be less than 5% for single
cell suspensions prepared from fresh/ frozen tissues, and less than 8% for
 74

nuclear suspension prepared from fixed, paraffin embedded specimens.

Where a diploid peak only is observed, one should ensure that tumor cells are
present in the clinical sample analyzed (Weinberg, 1996).

Full width at half maximum(c-a)

C.V. = 42.5× ‫ ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬%
 75

Peak (or mean) channel(b)

Figure (4-1): Coeffecient of Variation (C.V.) (Gustafson, 1982).

An aneuploid cell population was considered to be present when a

distinct peak was found constituting at least 2.5% of the total cell material
and deviating more than 10% from the diploid standard. DNA aneuploid is
reported when at least two separate G0/G1 peaks are demonstrated. For some
samples the diploid normal peak might be almost non existent; hence care
should be taken to assign peaks (Ormerod, 1994).

The degree of ploidy of this cell line was calculated by relating the
G1 maximum of these cells to DNA of the diploid G1 cells which are always
present. These diploid cells can be leukocytes, fibroblasts, normal urothelial
cells.

2 channel of aneuploid peak

Degree of ploidy= ————————————
Channel number of diploid peak

The degree of aneuploid was determined also by the DNA index

which represents the ratio of fluorescence intensity of aneuploid cells to the
diploid cells. The DNA index of a diploid tumor is 1.0, whereas, aneuploid
tumors are designated by progressively higher indices (Götte, 2001 and
Gorden et al., 2003).
 76

Estimation of the proportion of G1, S and G2+M cells made by

automatic integration of the cells in corresponding channels in the multi
channel analyzer. The values are corrected for background noise. To estimate
the proportions of cells in the S-phase of aneuploid tumor cell lines where
cells from the cell line coincide, the normal cell lines are subtracted.
Calculation of the phase distribution requires a minimum of about 1000 cells
combined with a low background noise resulting from cell fragments. These
calculations may also be rendered more difficult when the peaks for the
deploid or aneuploid cell population, partially or completely overlap. On the
other hand, there is no ambiguity in any of these cases to establish an
aneuploid cell line. An additional proof of the existence of an aneuploid
hyperdiploid cell line is the existence of a G2 peak occurs to the extreme
right of the histogram without interfering with the normal cell population
(Brown and Wittwer, 2000).

An aneuploid cell population with a tetraploid amount of DNA was

considered to exist when a peak exceeded the G2+ M peak found in normal
cells by three standard deviations or more. To quantitate the number of nuclei
normally found in the 4C or G2/ M peak, a number of control tissues were
studied. The mean percentage of nuclei in the 4C peak were 2.74+ 1.41
(standard deviation) for nuclei extracted from fresh normal lymphocyte.
These control data provide a firm basis for using greater than 10% of nuclei
in the 4C peak as a criterion for DNA polyploidy in the specimens
(Rabinovitch, 1994).

2- Immunophenotyping Applications:
The most common applications of flow cytometry are measurement
of DNA content in tumors and immunophenotyping of haematopoietic
 77

malignancies. Flow cytometry has shown to be a suitable method for

immunophenotyping of canine lymphomas (Culmsee and Nalte, 2002).

Immunophenotyping of abnormal cells is now considered a

fundamental tool to establish the cell lineage assignment and to obtain a more
precise identification of the various cell subtypes. Diagnostic
hematopathology depends on the applications of flow cytometric
immunophenotyping and immunohistochemical immunophenotyping
combined with the cytomorphology and histologic features of cases. The
availability of monoclonal antibodies directed against the surface proteins
permits flow cytometric analysis of erythrocytes, leukocytes and platelets
(Brown & Wittwer, 2000, Chianese, 2002 and Dunphy, 2004).

Multiparameter flow cytometry with optimally selected antibody

combinations has expanded the use of this technique beyond traditional
applications in hematopathology. By analyzing qualitative patterns of antigen
expression on discrete populations or "clusters," one can detect
immunophenotypic aberrancy in specific cell populations relative to normal
and reactive populations. Evaluation of patterns of antigen expression can
also be used to supplement conventional methodologies in the diagnosis and
subclassification of certain types of hematologic neoplasia. Finally, the
diagnosis of some congenital disorders affecting the hematolymphoid system
can be facilitated by the detection of characteristic immunophenotypic
changes (Kroft, 2004).

2-1-Erythrocyte analysis:
Tests that appear to have the greatest potential for routine
application of flow cytometry include reticulocyte and reticulated platelet
enumeration, detection of erythrocyte-bound immunoglobulin,
 78

immunophenotyping of leukemias and lymphomas, and bone marrow

differential cell counting (Brown and Wittwer, 2000 , Weiss, 2002).

Flow cytometric methods were first applied to laboratory

hematology with the improvement in reticulocyte counting and the creation
of the immature reticulocyte fraction for better anemia evaluation and
therapeutic monitoring (Davis, 2001).
 79

2-2-HIV monitoring:
More than 35 million people in developing countries are living with
HIV infection. While drug prices have dropped considerably, the cost and
technical complexity of laboratory tests essential for the management of HIV
disease, such as CD4 cell counts, remain prohibitive. New, simple, and
affordable methods for measuring CD4 cells that can be implemented in
resource-scarce settings are urgently needed (Dieye et al., 2005, Walker et
al., 2005 and Pattanapanyasat & Thakar, 2005).

2-3-Immunophenotyping of leukemias:
Immunophenotyping has become common in the diagnosis and
classification of acute leukemias and is particularly important in the proper
identification of cases of minimally differentiated acute myeloid leukemia.
To evaluate the immunophenotype of adult AML, cases were studied by
cytochemical analysis and by flow cytometry with a panel of antibodies
(Khalidi et al., 1998).

Characterization of leukemias by immunotyping is particularly

helpful when the morphology is difficult to interpret. The major advantage of
using immune markers by flow cytometry is the identification of particular
leukemia subtype, not recognized by morphologic criteria, which may have
prognostic significance (Rezaei et al., 2003).

Flow cytometric immunophenotypic analysis allowed to establish

diagnosis in cytomorphologically unclassified cases, identify acute mixed-
lineage leukemias (AMLL) with a frequency similar to that reported in other
series, and confirm the heterogeneity of acute leukemia (AL) (Piedras et al.,
1997 and Götte, 2001).
 80

Flow cytometry may be used to detect minimal residual disease

(MRD) in acute lymphoblastic leukemia because leukemic cells often display
aberrant phenotypes when compared to normal cells. Flow cytometry is a
sensitive and specific method for detecting MRD of childhood ALL, and
could predict the coming relapse (Zhang et al., 2005).

With the advent of monoclonal antibodies and a uniform

nomenclature system defining antibody reactivity in terms of clusters of
differentiation (CD), an independent means of characterizing acute leukemias
using cellular antigen expression has evolved. Immunophenotyping is usually
performed using immunofluoresence technique and is complementary to the
light microscopic based morphologic classification. This is especially true of
the lymphoid leukemias where morphology and cytochemistry cannot
distinguish among different lineage of lymphoid cells, such as B versus T
cells. With Immunophenotyping lineage is assigned using a panel of
monoclonal antibodies that identify the expression of cell surface antigens.
The panel of monoclonal reagents must include antibodies reactive with both
myeloid and lymphoid cells to distinguish between the two most important
groups. The reactivity pattern of the leukemia cells for all reagents is then
examined for the final assignment of lineage: B- lymphoid, T- lymphoid,
myeloid or undifferentiated (Maslak et al., 1994).

Comparative studies of cell surface antigen expression between

normal and leukemic cells indicate that most if not all leukemias express
phenotypes that are not observed in most normal maturing cells. This
aberrant expression of cellular antigens suggests that leukemias are not
proliferations of cells arrested at one state of normal maturation; rather
leukemic cells maintain a genetic program that can produce expression of
 81

antigens of any lineage. Nearly all laboratories performing

immunofluoresence analysis use different reagents (Terstappen et al., 1991).

2-4-Quantification of stem cells:

100 years ago, hematopoietic stem cells were postulated as blood
lymphocyte-like cells. Within the last 20 years, the frequency of autologous
and allogeneic transplantation of hematopoietic stem cells has increased.
Hematopoietic growth factors allow the stem cells' mobilization from the
bone marrow into the peripheral blood. Quantification of these hematopoietic
stem cells by means of flow cytometry can be achieved within hours
(Goldschmidt et al., 2003).

Flow cytometry has become the major technique for the quality
control of stem cell-containing products such as apheresis concentrates, bone
marrow or cord blood (Grieson et al., 1995). Stem cells can be easily
identified with flow cytometry due to their unique characteristics. They
demonstrate a medium level of CD34 expression, a low level of CD45
expression and a low forward side scattered (Jennings & Foon, 1997 and
Maslak et al., 1994).

2-5-Platelet analysis:
The analysis of platelets by flow cytometry is becoming more
common in both research and clinical laboratories. Platelet-associated
immunoglobulin assays by flow cytometry can be direct or indirect assays,
similar to other platelet-associated immunoglobulin immunoassays. In
autoimmune thrombocytopenic purpura, free serum antibodies are not found
as frequently as platelet-bound antibodies (Ashman et al., 2003).
 82

Immunofluorescent flow cytometry was used to measure the

percentage of activated platelet populations (CD62P, CD63), the percentage
of plt-monocyte aggregates (pma) (CD41/CD45), and activated monocytes
(CD11b, CD14, CD16) in the blood (Panasuik et al., 2005).

2-6-Testing for HLA-B27:

Human leukocyte antigen B27 (HLA-B27) is a major
histocompatibility complex class 1 molecule that is strongly associated with
the disease ankylosing spondylitis. The performance of the two flow
cytometric antigen assays depends on the antibody used and the positive
cutoff values assigned (Seipp et al., 2005). A flow cytometric HLA-B27 test
is much faster than the classical microcytoxicity test (Jennings and Foon,
1997 and Götte, 2001).

A biannual external quality assurance scheme for flow cytometric

typing of the HLA-B27 antigen is operational in The Netherlands and
Belgium since 1995. For flow cytometry, the most widely monoclonal
antibody used was FD705, followed by GS145.2 and ABC-m3. The majority
of laboratories used more than 1 anti-HLA-B27 monoclonal antibody for
typing (Seipp et al., 2005).

3- Major applications of apoptosis analysis:

There are many ways of detecting apoptosis by flow cytometry.

Apoptotic cells can be recognized by a characteristic pattern of
morphological (cell shrinkage, cell shape change, condensation of cytoplasm,
nuclear envelope changes, nuclear fragmentation, loss of cell surface
structures, apoptotic bodies, cell detachment and phagoctosis of remains),
 83

biochemical and molecular changes (free calcium ion rise, bcl2/Bax

interaction, cell dehydration, loss of mitochondrial membrane potential,
proteolysis, phosphatidylserine externalization, lamin B proteolysis, DNA
denaturatuin, 50-300kb cleavage, intranucleosomal cleavage and protein
cross-linking (Hubank et al., 2004 and Liu et al., 2004).

The methods of detecting apoptosis by flow cytometry are based on

the measurement of light scatter, the detection of changes in the plasma
membrane, the analysis of cell organelles or the sensitivity of DNA to
denaturation (Sedlak et al., 1999).

3-1-Apoptosis light scatter:

As cells die or become apoptotic the refractive index of the internal
cytoplasm becomes more similar to that of the extracellular medium this
manifests itself as a reduction in forward scatter signal. At the same time,
intracellular changes and invagination of the cytoplasmic membrane lead to
an increase in side (or orthogonal or 90º) scatter. If a dead cell discriminatory
dye is added, cells that have become permeable can be identifying.  In this
way low level resolution of dead and apoptotic cells can be get.  A number of
dead cell dyes are available for use and the one used will depend on any other
fluorochromes that are being measured.  Some examples are; Sytox Green
(488nm excitation; green fluorescence emission), Propidium Iodide (488nm
excitation; orange/red fluorescence emission), 7-Aminoactinomycin-D (7-
AAD) (488nm excitation; red fluorescence emission) and TO-PRO-3 (633nm
excitation; red fluorescence emission) (Cohen and Al-Rubeai, 1995).
 84

3-2-Apoptosis DNA analysis:

During apoptosis, calcium and magnesium dependent nucleases are
activated which degrade DNA. This means that within the DNA there are
nicks and fragmentation. We can detect these in three ways using DNA
analysis to look at the sub G1 peak, using strand break labeling (TUNEL) to
detect broken DNA or using Hoechst binding to detect DNA conformational
changes (Majino and Joris, 1995).

The sub-G1 Fig. (4-2) method relies on the fact that after DNA
fragmentation, there are small fragments of DNA that are able to be eluted
following washing in either PBS or a specific phosphate-citrate buffer. This
means that after staining with a quantitative DNA –binding dye, cells that
have lost DNA will take up less stain and will appear to the left of the G1
peak. The advantage of this method is that it is very rapid and will detect
cumulative apoptosis and is applicable to all cell types (Darzynkiewicz,
1997).

However in order to be seen in the sub G1 area, a cell must have lost
enough DNA to appear there, so if cells enter apoptosis from the S or G2/M
phase of the cell cycle or if there is an aneuploid population undergoing
apoptosis, they may not appear in the sub G1 peak (Schwartz and Osborne,
1993).
 85

Figure (4-2): Sub G1 peak by propidium iodide staining (Darzynkiewic,

1997).
 86

Also cells that have lost DNA for any other reason e.g. death by some
other form of oncosis, will appear in the sub G1 region so we have to be
careful about how we define the sub G1 peak (Nicoletti et al., 2001).

3-3-Apoptosis cell membrane analysis:

In normal cells, phosphatidylserine (PS) residues are found in the
inner membrane of the cytoplasmic membrane. During apoptosis, the PS
residues are translocated in the membrane and are externalized. In general
though not always, this is an early event in apoptosis and is though to be a
signal to neighboring cells that a cell is ready to be phagocytosed (Robinson,
1993).

Annexin-V is a specific PS-binding protein that can be used to detect

apoptotic cells. Annexin V- is available conjugated to a number of different
fluorochromes. Early apoptotic cells are annexin positive but PI negative.
Because the cells aren't fixed we can exclude dead cells and it is possible to
add further markers if the cytometer set up are appropriate. As with all live
cell assays, we have to remember that we are only looking at a snapshot of
the cells as they are at time of analysis and generally all apoptotic
experiments should be performed over a time course; Fig. (4-3) (Telford et
al., 2004, Homburg et al., 1995 and Vermes at al., 1995).
 87

Figure (4-3): Early apoptotic cells are annexin positive but (in this case)
PI (negative) (Telford et al., 2004).
 88

Hoechst 33342 is a DNA-binding dye that is able to quantitatively

stain the DNA of live cells.  However it has also been found that if the
concentration of Hoechst is low, the apoptotic cells take up the Hoechst more
rapidly.  If we also add PI or TO-PRO-3 we can specifically identify the dead
cells.  This is a rapid and quantitative method but requires the use of a UV
laser. The advantage of using TO-PRO-3 is that cell phenotyping using
FITC- and PE-labelled antibodies is also possible.  Thymocytes labelled with
CD4-PE and CD8-FITC can be assessed for apoptosis using Hoechst and
TO-PRO-3 (Koopman et al., 1994).

A third way of assessing the membrane changes in apoptosis is to use

YO-PRO-1 (Molecular Probes).  As this fluorochrome emits in the green, it
can be combined with propidium iodide to identify dead cells.  The rationale
here is that cells in early apoptosis are unable to pump out YO-PRO-1 but are
still not permeable to other dead cells discriminatory dyes (Koopman et al.,
1994).

3-4-Apoptosis enzyme analysis:

Two genes (ced-3 and ced-4) were crucial to the process of
apoptosis.  The ced-4 gene product has homologues in mammalian cells,
especially a family of cysteine proteases that are now known as caspases.
There are a number of caspases in mammalian cells that have been shown to
be involved in the early stages of apoptosis e.g. (caspase2, caspase3, caspase
6, caspase 9 and caspase 10). The functions of these enzymes are not yet
entirely clear but it appears that after an initial signal to the cell to undergo
apoptosis, they may be responsible for the activation, amplification and
execution of the apoptotic cascade (Cohen and Al- Rubeai, 1995).
 89

Because of the central importance of the caspases in apoptosis, their

detection by flow cytometry has become widespread.  We can detect the
activity of enzymes implicated in apoptosis in three ways; by detecting the
active form of the enzyme using a specific antibody (Smolewski et al., 2002),
by using a fluorochrome labelled peptide that binds to the active site of the
enzyme (Pozarowski et al., 2003) and by using a non-fluorescent substrate
for the enzyme which yields a fluorescent product if the enzyme is active
(Telford et al., 2004).

3-5-Apoptosis organelle analysis:

During apoptosis there is often a collapse of the mitochondrial
membrane potential. This can be detected in a number of ways by flow
cytometry. Two dyes in particular are useful- CMXRos (also known as Mito
tracker Red from Molecular probes) and LDs-751 (from Exction). CMXRos
has a chloromethyl group which allows accumulation in active mitochondria. 
Live cells that have active mitchondria are able to take up CMXRos but in
cells that are undergoing apoptosis, the mitochondrial membrane potential
decreases which means less dye accumulates in the mitchondria leading to a
decrease in fluorescenc (Chapman et al., 1995).

4- Detection of apoptotic markers:

Determination of p53 expression by immunohistochemistry (IHC)
has been incorporated into routine practice and its reliability has been
consolidated. However, flow cytometric (FCM) analysis might represent an
important objective and rapid approach. FCM may provide important
information about p53 protein expression in the different subpopulations and
cell cycle phases. In most breast, lung, and colon aneuploid tumors (77%),
 90

p53-positive cells were detected only in the subpopulations with abnormal

DNA content (Elvira et al., 1998).

Bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast

cell lines were analyzed via flow cytometry (FCM) for expression of p53 and
c-myc proteins along with their DNA content. At least 9,000-10,000 p53 or
c-myc protein molecules per cell were detected in the transformed
tumorigenic cell lines. These results show that quantitative FCM can be
reliably used to detect very low levels (3,000 molecules per cell) of specific
protein, and FCM is a useful tool to study the virus-induced changes in the
levels of nuclear proteins within a cell population and in tumorigenesis
(Agrawal et al., 1994).

In human follicular lymphoma, Analysis of transgenic Bcl2

expression used biotinylated Bcl2-100 monoclonal antibody for the surface
phenotyping of hematopoietic cells by flow cytometry. Cells (106 per analysis)
were stained with relevant antibodies labeled with fluorochromes (fluorescein
isothiocyanate [FITC], phycoerythrin [PE], or cyanin 5 [Cy5]) or biotin using
1% normal rat serum to block Fc receptors. Streptavidin conjugated to FITC
or PE was used as a secondary reagent for biotinylated antibodies. Analyses
were performed on a Life Sciences Research (LSR) or a FACStar II flow
cytometer (Becton Dickinson, San Jose, CA) (Alexander et al., 2004).

The expression of bcl-2 was examined by multicolor flow

cytometry in samples including lymph node or other tissue biopsy specimens
containing follicular lymphoma (FL), reactive hyperplasia (RH), or other
malignant lymphomas, as well as bone marrow aspirates. For all of the
aforementioned reasons, a reliable flow cytometric assay for expression of
 91

bcl-2 would be a useful additional technique for establishing a diagnosis of

FL. However, the measurement of bcl-2 by flow cytometric techniques has
received only scant attention. It was described a 2-color flow cytometric
assay using antibodies against bcl-2 that demonstrated promise in the
recognition of FL. (James et al., 2003).
 92

V-Flourescence in situ hybridization

1-Introduction:

FISH provides researchers with a way to visualize and map the

genetic material in an individual's cells, including specific genes or portions
of genes. This is important for understanding a variety of chromosomal
abnormalities and other genetic mutations. Unlike most other techniques used
to study chromosomes, FISH does not have to be performed on cells that are
actively dividing. This makes it a very versatile procedure. The first step is to
prepare short sequences of single-stranded DNA that match a portion of the
gene the researcher is looking for. These are called probes. The next step is to
label these probes by attaching one of a number of colors of fluorescent dye
(Schröck et al., 1996 and Fox et al., 1996).

DNA is composed of two strands of complementary molecules that

bind to each other like chemical magnets. Since the researchers' probes are
single-stranded, they are able to bind to the complementary strand of DNA,
wherever it may reside on a person's chromosomes. When a probe binds to a
chromosome, its fluorescent tag provides a way for researchers to see its
location (White et al., 1995 and Bloom, 2005).

Fluorescent in situ hybridization (FISH) represents a modem

molecular pathology technique, alternative to conventional cytogenetics
(karyotyping). Fluorescence in situ hybridization (FISH) allows identification
of specific sequences in a structurally preserved cell, in metaphase or
interphase (Kontogeorgos, 2004 and Celedaet al., 1994).
 93

The probe, bound to the target, will be developed into a fluorescent

signal. The fact that the signal can be detected clearly, even when fixed in
interphase, improves the accuracy of the results, since in some cases it is
extremely difficult to obtain mitotic samples Fig. (5-1) (Muhlmann, 2002).

The power of in situ hybridization can be greatly extended by the

simultaneous use of multiple fluorescent colors. Multicolor fluorescence in
situ hybridization (FISH), in its simplest form, can be used to identify as
many labeled features as there are different fluorophores used in the
hybridization. By using not only single colors, but also combinations of
colors, many more labeled features can be simultaneously detected in
individual cells using digital imaging microscopy (Raap et al., 1995).

Fluorescence, a phenomenon whereby a chemical excited at one

light wavelength emits light at a different and usually longer wavelength, is
used throughout the life sciences to study a wide variety of structures and
intracellular activities. Advances in probe and microscope technology have
led to the rapid development of techniques for fluorescence over the past
decade (Trask, 1991).

The accuracy of cytogenetic diagnosis in the management of

hematological malignancies has improved significantly over the past 10
years. Fluorescence in situ hybridization (FISH), a technique of molecular
cytogenetics, has played a pivotal role in the detection of unique sub-
microscopic chromosomal rearrangements that helped in the identification of
chromosomal loci, which contain genes involved in leukemogenesis (Amare
et al., 2001).
 94

Figure (5-1):

Fluoresence in situ hypridization (Muhlmann, 2002).

 95

The use of FISH is growing rapidly in genomics, cytogenetics,

prenatal research, tumor biology, radiation labels, gene mapping, gene
amplification, and basic biomedical research. In principle, the technique is
quite straightforward (Attarbaschi et al., 2004).

The hybridization reaction identifies, or labels, target genomic

sequences so their location and size can be studied. DNA or RNA sequences
from appropriate, chromosome-specific probes are first labeled with reporter
molecules, which are later identified through fluorescence microscopy. The
labeled DNA or RNA probe is then hybridized to the metaphase
chromosomes or interphase nuclei on a slide. After washing and signal
amplification, the specimen is screened for the reporter molecules by
fluorescence microscopy (Hohman and Gundlach, 1994).

FISH probes are commonly used to detect the presence of specific

DNA sequences either when DNA is condensed into metaphase
chromosomes or dispersed in non dividing interphase cells. The fact that
hybridization of probes to metaphase chromosomes is visualized in two
dimensions while interphase targets are three dimensional has implications
for both validations of assays and the development of baseline reference
ranges (Pauletti et al., 1996).

Metaphase applications generally yield clear yes/no answers while

interphase applications commonly require reportable reference ranges before
interpreting of results. In addition to determining the presence or absence of
particular sequences in the genome, FISH is useful in assessing gene copy
number in some disorders (Massod et al., 1998).
 96

Analytical uncertainty over DNA probe assays also may stem from
issues related to inherent population variation. The use of some repeat
sequence probes has been discontinued because of inability to detect targeted
sequences in individuals who possess very few repeats, leading to insufficient
probe label in the targeted region which precludes visualized of the signal.
Such probes have been eliminated (Myrata et al., 1997 and Bossuyt et al.,
1995).

FISH allows very precise spatial resolution of morphological and

genomic structures. The technique is rapid, simple to implement, and offers
great probe stability. The genome of a particular species, entire
chromosomes, chromosomal-specific regions, or single-copy unique
sequences can be identified, depending on the probes used (Attarbaschi et
al., 2004).

Until recently, FISH was limited by the hardware, software,

reagents, probe technology, and cost involved in implementing the technique.
Commercially available microscope hardware optimized for multicolor FISH
was not available until the mid-1990s. Prior to that, microscopes had to be
customized for FISH applications. Most microscope optics was not designed
to detect the low light levels inherent in FISH signals. As the genomic
resolution of the technique has increased dramatically, the requirements on
microscope optics have further increased. Chromatic aberrations among
multiple wavelengths have been a problem. For multicolor analysis in
particular, all lenses, including the collector lens, had to be chromatically
corrected. In addition, epi-fluorescence light sources were difficult to align
for uniform illumination (Amare et al., 2001).
 97

Analysis of multicolor FISH images requires isolation of the various

signals either with individual filter cubes; or utilization of an excitation filter
wheel with multipass dichroic and barrier filters. Recent developments in
filter technology corrected some of the previous problems encountered
through optical misalignments caused by mechanical switching of individual
filter cubes. Excitation filter wheels used with multi-pass dichroic and barrier
filters can be used effectively for up to three colors by employing separate
excitation filters for each color with no registration shift. But, for more than
three colors, single-pass filters still had to be used (Racevskis, 2005,
Iarovaia et al., 2005 and Iourov et al., 2005).

2-Three different types of FISH probes:

2-1-Locus specific probes

They bind to a particular region of a chromosome. This type of

probe is useful when scientists have isolated a small portion of a gene and
want to determine on which chromosome the gene is located (Hjalmar, 2005
and Wang et al., 2005).

2-2-Alphoid or centromeric repeat probes

They are generated from repetitive sequences found in the middle of
each chromosome. Researchers use these probes to determine whether an
individual has the correct number of chromosomes. These probes can also be
used in combination with "locus specific probes" to determine whether an
individual is missing genetic material from a particular chromosome
(Edward et al., 2005).
 98

2-3-Whole chromosome probes

They are actually collections of smaller probes, each of which binds

to a different sequence along the length of a given chromosome. Using
multiple probes labeled with a mixture of different fluorescent dyes,
scientists are able to label each chromosome in its own unique color. The
resulting full-color map of the chromosome is known as a spectral karyotype.
Whole chromosome probes are particularly useful for examining
chromosomal abnormalities, for example, when a piece of one chromosome
is attached to the end of another chromosome (Dugan et al., 2005).

3-Applications of FISH:
The clinical uses of FISH were considered in three main areas;
diagnosis of individuals with birth defects and mental retardation, prenatal
diagnosis and screening, and identification and monitoring of acquired
chromosome abnormalities in leukemia/ cancer. In each area the critical
consideration remains a clear understanding of the capabilities and
limitations of a test to provide useful information (Bossuyt et al., 1995 and
Pauletti et al., 1996).

Traditional cytogenetic analysis, detecting deletions, duplications,

rearrangement and the identifications of unknown material of marker or
derivative chromosomes, in individuals with birth defects and/or mental
retardation has led to an understanding of the etiology of a number of
syndromes. The clinical utility and limitations of these tests are both general
and disease specific (Ledbeteer et al., 1987, Callen et al., 1992, Ribeiro et
al., 1997 and Cassidy et al., 1998).
 99

Prenatal applications of FISH testing include both screening tests

and diagnostic tests. Technical issues are few, and clinical utility raises
questions as to the intended use of FISH in testing. The application of FISH
to prenatal screening for common autosomal trisomies and sex chromosome
anomalies is becoming increasingly common. The primary considerations
involve differing clinical sensitivity between the abnormalities detected by
classical cytogenetic versus these detected by FISH based assays (Evans et
al., 1991 and Klinger et al., 1992).

Among cases ascertained via ultrasonographically identified fetal

anomalies, some may be conclusive for a syndromes diagnosis and may be
approached by a (diagnostic) FISH test. Families in which subtle or
submicroscopic chromosomal abnormalities, detectable by FISH, are known
to segregate will benefit greatly from prenatal FISH studies (Kontogeorgos et
al., 2000 and Lewin et al., 2000).

Fluorescence in situ hybridization (FISH) has become one of the

major techniques in environmental microbiology. The original version of this
technique often suffered from limited sensitivity due to low target copy
number or target inaccessibility (Zwirglmaier, 2005).

The reagents and probes themselves were not sufficient for all
applications. For instance, the efficiency of hybridization site detection
decreased with decreasing probe size, creating significant limits to what
could be observed via fluorescence microscopy. The number of differently
colored fluorescent dyes was limited, and the photostability of the dyes was
poor. But new developments in fluorescent dye technology and spin-off
technology from the federally funded Human Genome Project are now
having an impact. There are probes for all the human chromosomes and a
 100

growing number of new gene-specific probes are available. In situ

hybridization kits and fluorescently labeled probes are commercially
available from several companies (Sarrate et al., 2005).

The ability of FISH to rapidly test interphase and metaphase

chromosome defects makes it especially useful in the study of cancer. In
solid tumors, conventional cytogenetics is rarely used because obtaining
metaphases is difficult and those cells that do proceed to mitosis may not be
representative of the tumor. Other molecular techniques, such as PCR and
Southern, Northern, and Western analysis, require extraction of the tissue.
Extraction procedures net both normal and abnormal cells, so sensitivity is
lower and quantitation less reliable than with FISH probes (Bosch et al.,
2005).

FISH allows cell-by-cell analysis and thus provides for a more

sensitive and reliable assessment of chromosomal aneuploidy, gene
amplifications and deletions, and chromosome translocations. A reliable
determination of whether a gene is amplified in a specimen is often possible
with evaluation of only 20 to 50 cells (Ogilvie et al., 2005).

The accuracy of cytogenetic diagnosis in the management of

hematological malignancies has improved significantly over the past 10
years. FISH has played a pivotal role in the detection of unique sub-
microscopic chromosomal rearrangements that helped in the identification of
chromosomal loci, which contain genes involved in leukemogenesis (Amare
et al., 2001).

FISH was performed with specific probes to make the rapid prenatal
diagnosis of Down syndrome. FISH was performed respectively with locus-
 101

specific probe (LSI) and centromeric probe (CEP) X/Y on the uncultured
amniotic fluid. FISH is a rapid and reliable method to detect Down syndrome
in uncultured amniotic fluid (Wang et al., 2005)

Fluorescence in situ hybridization assay and to correlate the genetic

findings with the pathologic grade and stage were used to investigate the
chromosomal abnormalities present in bladder carcinoma (Placer et al.,
2005).

A novel application of FISH to isolated nuclei is described. The

method detects gene amplification and chromosome aneuploidy in extracted
nuclei from paraffin-embedded tissue of human cancer with greater
sensitivity and specificity than existing FISH methods. The method is applied
to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is
one of the most common genetic alterations associated with human breast
cancer (Rossi et al., 2005).

Tumor-specific chromosomal abnormalities are attracting a large

interest owing to the diagnostic, prognostic, and therapeutic importance. The
development of FISH has improved the detection of specific chromosomal
abnormalities in chronic lymphocytic leukemic (CLL). By using FISH, the
problem with tumor cells with low mitotic rate is avoided since this method
readily detects clonal aberrations also in nondividing, interphase cells. Three
different types of probes are used centromeric probes for numerical
chromosome abnormalities, whole chromosome paints, and locus-specific
probes for numerical chromosome abnormalities, whole chromosome paints,
and locus-specific probes. (Hjalmar, 2005)
 102

FISH of DNA-DNA or DNA-RNA using post-mortem brain samples

is one approach to study low-level chromosomal aneuploidy and selective
expression of specific genes in the brain of patients with neuropsychiatric
diseases. FISH could be applied to extended studies of chromosomal
aneuploidy, abnormal patterns of chromosomal organization and functional
gene expression in situ in the neurons of the brain in different psychiatric and
neurodevelopmental diseases (Yurov et al, 2001).

3-1-ALL investigation by FISH:

To investigate patients with acute lymphoblastic leukemia (ALL) for
TEL/AML1 fusion, BCR/ABL fusion, MLL gene rearrangements, and
numerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situ
hybridization (FISH) and to determine the relationship and the significance of
those findings (Zhang et al., 2003).

Interphase fluorescence in situ hybridization (iFISH) is increasingly

used for the identification of BCR/ABL gene rearrangements in chronic
myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). FISH
plays an important role in detecting chromosome changes, especially in some
cryptic chromosome translocations and patients with culture failures (Primo
et al., 2003 and Zhang et al., 2003).

ALL blasts routinely contain somatically acquired genetic

abnormalities that provide insight into pathogenesis and strongly influence
prognosis. Approximately one third of cases of ALL show an increase in the
modal chromosome number (e.g., hyperdiploid, > 47 chromosomes, and
"high" hyperdiploid, > 50 chromosomes) blasts make up a unique biologic
subset associated with increased in vitro apoptosis and sensitivity to a variety
 103

of chemotherapeutic agents (Heerema et al., 2000 and Trueworthy et al.,

1992).

Almost one third of ALL blasts show chromosomal translocations in

the absence of changes in chromosome number. Four major translocations
have been observed, and each defines a unique biological subset of patients.
The t(1;19)(q23;q13) is a hallmark of some pre-B (cytoplasmic µ+) ALL, and
is characterized by fusion of the E2A and PBX genes (Uckun et al., 1998).

Despite the adverse prognostic impact of this translocation in older

studies, recent intensification of therapy has resulted in an improved survival
for these children. Translocations between the mixed lineage leukemia (MLL)
gene at 11q23 and over 30 different partner chromosomes characterize 6% of
ALL cases. MLL translocations, most commonly t(4;11)(q21;q23), are seen in
the vast majority of infant patients with ALL. A recent, large series
demonstrates that any rearrangement of 11q23 is associated with a worse
prognosis (e.g., 20% to 25%) (Pui et al., 2002).

3-1-1-Philadelphia
The presence of the t(9;22)(q34;q11) translocation, commonly
known as Philadelphia chromosome (Ph), in about 3% to 5% of all children
with ALL is considered as one of the molecular markers associated with a
particularly high risk for treatment failure (Ribeiro et al., 1987, Crist et
al.,1990, Pui et al., 1990, Fletcher et al., 1991, Reiter et al., 1994, and
Chessells et al., 1995) .

This translocation causes a rearrangement between the

protooncogene c-ABL and a gene called the breakpoint cluster region (BCR).
Whereas the breaks in c-ABL occur mainly in the same region (between the
 104

exons a1 and a2) on chromosome 9, two different ones affect the breakpoint
cluster region on chromosome 22: the more frequent one (approximately in
2 of 3 of all cases) shows a break in the minor breakpoint cluster region (m-
BCR) between the exons e1 and e2. This is predominant in ALL. In 1 of 3 of
all Ph+ ALL cases, the major (M-) BCR found between exons b2 and b3 or
exons b3 and b4 is affected. M-BCR is also found in nearly all patients with
chronic myelogenous leukemia (CML). Chimeric proteins of 210 kD (p210)
and 190 kD (p190) result from the M-BCR/ABL and m-BCR/ABL
rearrangements, respectively (Kantarjian et al., 1991).

These fusion proteins cause a deregulation of protein tyrosine kinase

activity. Both forms of the chimeric gene (BCR/ABL) can be detected by
polymerase chain reaction (PCR) and fluorescent in situ hybridization.
(Maurer et al., 1991, Dewald et al., 1993, Schlieben et al., 1996).

Most patients with Philadelphia (Ph)-positive acute lymphoblastic

leukemia (ALL) show evidence of secondary chromosome aberrations that
may influence the course of disease and response to treatment. To better
understand how these secondary chromosomal aberrations occur and to
investigate whether the p185/p190 BCR-ABL fusion protein may directly
induce an increased chromosomal instability and subsequently the
appearance of clonal chromosome aberrations, three BRC-ABL (p185/
p190)-transduced mouse pre-B cell lines were analyzed by spectral
karyotyping and fluorescence in situ hybridization. The human wild-type
BCR-ABL gene was expressed at a level comparable with that in human Ph-
positive leukemias at diagnosis. All BCR-ABL-transduced cell lines acquired
similar clonal chromosomal aberrations. Trisomy 5 was always present,
followed by loss of the Y chromosome, trisomy of chromosomes 12 and 18,
and an unbalanced translocation between chromosomes X and 12. Thus,
 105

ectopic p185/p190 BCR-ABL expression, such as p210 BCR-ABL, PML-

RARA, or C-MYC transduction, may induce an increased chromosomal
instability leading to clonal karyotypic evolution, which may mimic
secondary chromosome aberrations in human Ph-positive ALL (Rudolph et
al., 2005).

The Philadelphia (Ph) chromosome, the main product of the (9;22)

(q34;q11) translocation, is the cytogenetic hallmark of chronic myeloid
leukemia (CML), a clonal myeloproliferative disorder of the hematopoietic
stem cell; the Ph chromosome is also found in a sizeable portion of acute
lymphoblastic leukemia (ALL) patients and in a small number of acute
myeloid leukemia (AML) cases. Three different breakpoint cluster regions
are discerned within the BCR gene on chromosome 22: M-bcr, m-bcr, and
mu-bcr (Drexler et al., 1999).

Nearly all Ph + ALL cell lines have the m-bcr e1-a2 fusion gene
(only two ALL cell lines have a b3-a2 fusion) whereas all CML cell lines, but
one carry the M-bcr b2-a2, b3-a2 or both hybrids. The mu-bcr e19-a2 has
been detected in one CML cell line. Four cell lines display a three-way
translocation involving chromosomes 9, 22 and a third chromosome.
Additional Ph chromosomes (up to five) have been found in four Ph + ALL
cell lines and in 18 CML cell lines; though in some cell lines the extra Ph
chromosome(s) might be caused by the polyploidy (tri- and tetraploidy) of
the cells. Another modus to acquire additional copies of the BCR-ABL fusion
gene is the formation of tandem repeats of the BCR-ABL hybrid as seen in
CML cell line K-562. Both mechanisms, selective multiplication of the
der(22) chromosome and tandem replication of the fusion gene BCR-ABL,
presumably lead to enhanced levels of the fusion protein and its tyrosine
kinase activity (genetic dosage effect). The availability of a panel of Ph + cell
 106

lines as highly informative leukemia models offers the unique opportunity to

analyze the pathobiology of these malignancies and the role of the Ph
chromosome in leukemogenesis (Drexler et al., 1999).

Treated children with acute lymphoblastic leukemia were analysed

for chromosomal abnormalities with conventional G-banding, spectral
karyotyping (SKY) and interphase fluorescent in situ hybridisation (FISH)
using probes to detect MLL, BCR/ABL, TEL/AML1 rearrangements and
INK4 locus deletions. Three novel TEL partner breakpoints on 1q41, 8q24
and 21p12 were identified, and a recurrent translocation t(1;12)(p32;p13) was
found. In addition, two cases displayed amplification (7-15 copies) of AML1.
Results were demonstrated the usefulness of SKY and interphase FISH for
the identification of novel chromosome aberrations and cytogenetic
abnormalities that provide prognostically important information in childhood
ALL (Nordgren et al., 2002).

The BCR/ABL and MLL/AF4 fusion genes--resulting from t(9;22)

(q34;q11) and t(4;11)(q21;q23) translocations, respectively--are considered
as a high risk prognostic factors in children with acute lymphoblastic
leukaemia (ALL). Their presence in malignant cells indicates patient for the
most intensive antileukaemic therapy regardless of the other criteria. In
contrast, the most common non-random chromosomal aberration in
paediatric ALL--translocation t(12;21)(q12;q22)--is associated with a
favourable prognosis. The examination of these rearrangements is important
for the stratification of patients to the risk groups and also provides the most
sensitive and specific tool for minimal residual disease (MRD) follow-up
(Trka et al., 1999 and Poplack, 1993).
 107

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 145

VII- LIFE FLOWCYTOMETRIC FIGURES

(A) (B)

M2
M1

Figure (1): Flow cytometric analysis of c-myc expression on

mononuclear cells showing diagram (A) and dot plot (B) of positively
stained cells in relation to negative ones.

(A) (B)

R2

M2

M1
R1

Figure (2): Flowcytometric analysis of p53 expression on mononuclear

cells showing histogram (A) and dot plot (B) of positively stained cells in
relation to negative ones.
 146

Diploid: 100.00% Diploid: 100.00%

Dip G0-G1: 93.93 % at 33.49 Dip G0-G1: 90.43 % at 35.31
Dip G2-M: 4.57 % at 61.69 Dip G2-M: 7.84 % at 65.30
Dip S: 1.50 % G2/G1: 1.84 Dip S: 1.73 % G2/G1: 1.85
Dip %CV: 2.84 Dip %CV: 3.17

Figure (3): Histogram showing cell cycle parameters (diploid) using flow
.cytometer FACS caliber program modfit
 147

Diploid: 85.25 % % Diploid: 62.08

Dip G0-G1: 100.00 % at 38.52 Dip G0-G1: 100.00 at33.10
Dip G2-M: 0.00 % at 77.05 Dip G2-M: 0.00 % at 66.21
Dip S: 0.00 % G2/G1: 2.00 Dip S: 0.00 % G2/G1:2.00
Dip %CV: 3.42 Dip %CV: 3.16
% Aneuploid 1: 14.75 % Aneuploid 1: 37.92
Aneup G0-G1: 65.14 % at 69.75 Aneup G0-G1: 95.37 % at45.19
Aneup G2-M: 28.72 % at 104.73 Aneup G2-M: 1.76 % at 73.95
Aneup S: 6.13 % G2/G1: 1.50 Aneup S: 2.88 % G2/G1: 1.64
Aneup %CV: 3.31 Aneup %CV: 3.43
Aneup DI: 1.81 Aneup DI: 1.37

Figure (4): Histogram showing cell cycle parameters diploid and

aneuploid using flow cytometer FACS caliber program modfit.
 148

VIII- LIFE FISH PICTURES

Figure A Figure B

Figure C Figure D

Figure (A,B,C and D): Each childhood acute lymphoblastic leukemia

case shows red signal which is ABL on chromosome 9 and green signal
which is the breakpoint cluster region (BCR) on chromosome 22 for
children with Philadelphia negative acute lymphoblastic leukemia (Ph‾
ALL).
 149

Figure E Figure F

Figure (E and F): Each childhood acute lymphoblastic leukemia case

shows red signal which is ABL on chromosome 9, green signal which is
the breakpoint cluster region (BCR) on chromosome 22 and pale orange
signal which is the fusion (BCR/ABL) for children with Philadelphia
positive acute lymphoblastic leukemia (Ph+ ALL).